Compositions and methods for measuring blood glucose levels

ABSTRACT

In some embodiments, the present invention a mutated FAD-GDHa protein, wherein the mutated FAD-GDHa protein is mutated from a wild-type first species to contain at least one point mutation, wherein the mutated FAD-GDHα protein comprises: P(X)n=8X4(X)n=16V(X)n=6RN(X)n=3YDXRPXCXGX3NNCMP(X)n=1CP(X)n=2A(X)n=1Y(X)n=1G(X)n=6A(X)n=2AG(X)n=6AVV(X)n=3E(X)n=8-9A(X)n=2Y(X)n=1D(X)n=5HRV(X)n=5V(X)n=2A(X)n=3E(X)n=2K(X)n=4S(X)n=5P(X)n=1G(X)n=2N(X)n=4GRN(X)n=1MDH(X)n=4V(X)n=1F(Xn=6-7W(X)n=1GRGP(X)n=9RDGXX5R(X)n=19T(X)n=14L(X)n=14X2(X)n=1X1(X)n=1E(X)n=4P(X)n=1NR(X)n=3S(X)n=4D(X)n=2G(X)n=7Y(X)n=4Y(X)n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X1 is selected from the group consisting of X, S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X1 is L, H or V, then X2 is D; b) X3 is selected from the group consisting of G, H, D, Y, S, and X; c) X4 is selected from the group consisting of S and X; and d) X5 is selected from the group consisting of S and X.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 62/195,900, entitled “Glucose Dehydrogenase and Methods of UseThereof”, filed on Jul. 23, 2015; 62/195,914, entitled “GlucoseDehydrogenase and Methods of Use Thereof”, filed on Jul. 23, 2015; and62/345,386, entitled “Compositions and Methods for Measuring BloodGlucose Levels”, filed on Jun. 3, 2016, the contents of which are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

In some embodiments, the instant invention is related to compositionsand methods for measuring blood glucose levels.

BACKGROUND

Blood glucose monitoring is a way of testing the concentration ofglucose in the blood (glycemia). Particularly important in the care ofdiabetes mellitus, a blood glucose test is performed by piercing theskin (typically, on the finger) to draw blood, then applying the bloodto a chemically active disposable ‘test-strip’. The test is usuallyreferred to as capillary blood glucose. Current teaching counselsdiabetic patients to measure their blood glucose level from two to seventimes a day depending on the nature and severity of their individualcases. Based on the observed pattern in the measured glucose levels, thepatient and physician together make adjustments in diet, exercise andinsulin intake to better manage the disease. This information should beavailable to the patient immediately.

A biosensor is a sensor which utilizes the molecule identifyingabilities of biological materials such as microorganisms, enzymes, andantibodies to apply the biological materials as molecule recognitionelements. To be specific, the biosensor utilizes a reaction which occurswhen an immobilized biological material recognizes a target specificcomponent, such as oxygen consumption by respiration of amicro-organism, an enzyme reaction, or luminescence. Among biosensors,enzyme sensors have been advanced in practical applications, and forexample, enzyme sensors for glucose reduces an electron acceptor by anelectron generated by a reaction between an enzyme and a substrateincluded in a sample solution as a specimen, and a measurement deviceelectrochemically measures the oxidation-reduction quantity of theelectron acceptor, thereby to perform quantitative analysis of thespecimen. The first electrochemical glucose biosensor relied on a thinlayer of glucose oxidase (GO_(X)) entrapped over an oxygen electrode viaa semipermeable dialysis membrane. Measurements were made based on themonitoring of the oxygen consumed by the enzyme-catalyzed reaction (Wang2008). Second and third generation biosensors also rely on the effectsof enzyme-catalyzed reactions to determine glucose levels (Ferri et al.2011).

SUMMARY OF INVENTION

In one embodiment, the present invention is a mutated FAD-GDHα protein,wherein the mutated FAD-GDHα protein is mutated from a wild-type firstspecies to contain at least one point mutation, wherein the mutatedFAD-GDHα protein comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of X, S, C, T, M, V,        Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or        V, then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is selected from the group consisting of S and X; and    -   d) X⁵ is selected from the group consisting of S and X.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is selected from the group consisting of S and X; and    -   d) X⁵ is selected from the group consisting of S and X.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In one embodiment,

-   -   a) X¹ is X;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is S; and    -   d) X⁵ is X.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is X; and    -   d) X⁵ is S.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is X;    -   c) X⁴ is S; and    -   d) X⁵ is S.

In one embodiment,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is S; and    -   d) X⁵ is S.

In one embodiment, the amino acid sequence of the mutated FAD-GDHαprotein comprises the amino acid sequence set forth in any one of SEQ IDNOS: 9-29, SEQ ID NOS: 46-104, or SEQ ID NOS: 105-129.

In one embodiment, a biochemical activity is increased at least 10%compared to a non-mutated FAD-GDHα protein from the wild type firstspecies.

In one embodiment, a selectivity for glucose is increased at least 10%compared to a non-mutated FAD-GDHα protein from the wild type firstspecies.

In one embodiment, a linearity of current as a function of glucoseconcentration is increased at least 10% compared to a non-mutatedFAD-GDHα protein from the wild type first species.

In one embodiment, the present invention provides an enzyme electrode,configured to measure the amount of glucose in a physiological fluid,comprising the mutated FAD-GDHα protein according to some embodiments ofthe present invention immobilized onto the electrode, wherein themutated FAD-GDHα protein according to some embodiments of the presentinvention is configured to catalyze glucose in the physiological fluidand produce electrons that are transferred to the electrode therebygenerating an electrical current, wherein the intensity of theelectrical current is indicative of the level of glucose in thephysiological fluid.

In one embodiment, the enzyme electrode is a screen printed electrode.

In one embodiment, the enzyme electrode is configured to perform asingle measurement.

In one embodiment, the enzyme electrode is incorporated into a glucosetest strip.

In one embodiment, the mutated FAD-GDHα protein is immobilized on theelectrode in a conductive matrix.

In one embodiment, the conductive matrix is selected from a groupconsisting of carbon paste, graphite paste, graphene oxide, or any otherconductive matrix paste appropriate for use in screen printedelectrodes.

In one embodiment, the conductive matrix is a conductive polymer.

In one embodiment, the conductive polymer is selected from the groupconsisting of: PEDOT, and Polypyrrol.

In one embodiment, the conductive polymer is electro-polymerized on theelectrode together with the mutated FAD-GDHa protein.

In one embodiment, the conductive polymer is chemically polymerized onthe electrode together with the mutated FAD-GDHa protein.

In one embodiment, the mutated FAD-GDHα protein of the present inventionis immobilized to the electrode by chemical wiring.

In one embodiment, the conductive matrix further comprises an electronmediator.

In one embodiment, the enzyme electrode, configured to measure theamount of glucose in a physiological fluid, comprising the mutatedFAD-GDHa protein according to some embodiments of the present inventionimmobilized onto the electrode further comprises at least one subunitselected from the group consisting of: wild-type FAD-GDHβ subunit, and awild-type FAD-GDHγ subunit.

In one embodiment, the enzyme electrode of the present invention isincorporated into a biosensor configured for subcutaneous continuousglucose measurement, wherein the biosensor is configured to continuallymeasure the amount of glucose in the subject.

In one embodiment, the biosensor is configured to continuously measurefor up to two weeks.

In one embodiment, the biosensor comprises the mutated FAD-GDHα proteinaccording to some embodiments of the present invention immobilized ontoat least one enzyme electrode, wherein the mutated FAD-GDHα proteinaccording to some embodiments of the present invention is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current, whereinthe intensity of the electrical current is indicative of the level ofglucose in the subject.

In one embodiment, the electrode is made from a material selected fromthe group consisting of: carbon fiber, graphite, glassy carbon, gold,silver, copper, platinum, palladium, and metal oxide.

In one embodiment, the metal oxide is indium tin oxide.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be further explained with reference to theattached drawings, wherein like structures are referred to by likenumerals throughout the several views. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the present invention. Further, somefeatures may be exaggerated to show details of particular components.

FIGS. 1A and 1B show some aspects of some embodiments of the presentinvention. FIG. 1B shows the biochemical response of FAD-GDHα F406L tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 1B shows the biochemical response of F406L to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.

FIG. 2 shows some aspects of an embodiment of the composition of thepresent invention, showing the electrochemical data in connection withFAD-GDHα F406L.

FIGS. 3A-3C show some aspects of some embodiments of the presentinvention. FIG. 3A shows the biochemical response of FAD-GDHα F406A tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 3B shows the biochemical response of F406A to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406A enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 3C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 4A-C show some aspects of some embodiments of the presentinvention. FIG. 4A shows the biochemical response of FAD-GDHα F406C tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 4B shows the iochemical response of F406C to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406C enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 4C shows theelectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 5A-C shows aspects of some embodiments of the present invention.FIG. 5A shows the biochemical response of FAD-GDHα F406E to varyingconcentrations of glucose (rhombus), and xylose (rectangle). FIG. 5Bshows the biochemical response of F406E to glucose and the non-linearfit through which K_(m) (k) and V_(max) have been obtained. F406E enzymeactivity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 5C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 6A-C show some aspects of some embodiments of the presentinvention. FIG. 6A shows the biochemical response of FAD-GDHα F406D tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 6B shows the biochemical response of F406D to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406D enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 6C shows theelectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 7A-C show some aspects of some embodiments of the presentinvention. FIG. 7A shows the biochemical response of FAD-GDHα F406G tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 7B shows the biochemical response of F406G to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406G enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 7C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 8A and 8B show aspects of some embodiments of the presentinvention. FIG. 8A shows the biochemical response of FAD-GDHα F406H tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 8B shows the biochemical response of F406H to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.

FIGS. 9A-C show some aspects of some embodiments of the presentinvention. FIG. 9A shows the biochemical response of FAD-GDHα F406I tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 9B shows the biochemical response of F406I to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406I enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 9C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 10A and 10B show some aspects of some embodiments of the presentinvention. FIG. 10A shows the biochemical response of FAD-GDHα F406M tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 10B shows the biochemical response of F406M to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406M enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀.

FIGS. 11A-C show some aspects of some embodiments of the presentinvention. FIG. 11A shows the biochemical response of FAD-GDHα F406N tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 11B shows the biochemical response of F406N to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406N enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 11C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 12A and 12B show some aspects of some embodiments of the presentinvention. FIG. 12A shows the biochemical response of FAD-GDHα F406Q tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 12B shows the biochemical response of F406Q to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.

FIGS. 13A-C show some aspects of some embodiments of the presentinvention. FIG. 13A shows biochemical response of FAD-GDHα F406S tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 13B shows biochemical response of F406S to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406S enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 13C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 14A-C show some aspects of some embodiments of the presentinvention. FIG. 14A shows the biochemical response of FAD-GDH]α F406T tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 14B shows biochemical response of F406T to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406T enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 14C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 15A-B show negative results. FIG. 15A shows a biochemical responseof FAD-GDHα F406W to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 15B shows a biochemical response of F406W toglucose and the non-linear fit through which K_(m) (k) and V_(max) havebeen obtained.

FIGS. 16A-C show some aspects of some embodiments of the presentinvention. FIG. 16A shows the biochemical response of FAD-GDHα F406Y tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 16B shows the biochemical response of F406Y to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406Y enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 16C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 17A-C show some aspects of some embodiments of the presentinvention. FIG. 17A shows the biochemical response of FAD-GDHα F406V tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 17B shows the biochemical response of F406V to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406V enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 17C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 18A-C show some aspects of some embodiments of the presentinvention. FIG. 18A shows the biochemical response of FAD-GDHα F406P tovarying concentrations of glucose (rhombus), and xylose (rectangle).FIG. 18B shows the biochemical response of F406P to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been obtained.F406P enzyme activity was determined via monitoring decrease ofDichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 18C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

FIGS. 19A-B show some aspects of some embodiments of the presentinvention. FIG. 19A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215G to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 19B shows the non-linearfit of the data shown in FIG. 19A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 20A-B show some aspects of some embodiments of the presentinvention. FIG. 20A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215H to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 20B shows the non-linearfit of the data shown in FIG. 20A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 21A-B show some aspects of some embodiments of the presentinvention. FIG. 21A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215T to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 21B shows the non-linearfit of the data shown in FIG. 21A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 22A-B show some aspects of some embodiments of the presentinvention. FIG. 22A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215D to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 22B shows the non-linearfit of the data shown in FIG. 22A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 23A-B show some aspects of some embodiments of the presentinvention. FIG. 23A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215Y to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 23B shows the non-linearfit of the data shown in FIG. 23A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 24A-B show some aspects of some embodiments of the presentinvention. FIG. 24A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215S to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 24B shows the non-linearfit of the data shown in FIG. 24A, from which K_(m) (k) and V_(max) havebeen calculated.

FIG. 25A shows electrochemical data representing the current response ofthe biosensor to various glucose concentrations (shown as a rhombus),maltose concentrations (shown as a triangle) and xylose (shown as arectangle) concentrations (substrate solution were supplemented with 1mM benzoquinone). R² represents a linear fit of the glucose data. FIG.25B shows a biosensor response to glucose and the non-linear fit throughwhich apparent K_(m) (k) and I_(max) have been calculated based onMichaelis-Menten

$\begin{matrix}{I = \frac{{Imax}\lbrack S\rbrack}{{Km} + \lbrack S\rbrack}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Wherein “I” is the current, S is the substrate concentration, I_(max) isthe maximum current and the K_(m) is the apparent Michaelis constant.

FIG. 26A shows an exemplary embodiment of a bioelectrochemical responseof an FAD-GDHα mutant (N177S, N215S, F353L, F406L) of the presentinvention, varying concentrations of glucose (shown as a rhombus). FIG.26B shows a bioelectrochemical response of an FAD-GDHα mutant (N177S,N215S, F353L, F406L) of the present invention to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been calculated.The enzyme activity of FAD-GDHα (N177S, N215S, F353L, F406L) wasdetermined by immobilizing FAD-GDHα (N177S, N215S, F353L, F406L) to acarbon electrode by an electropolymerization method without the additionof an electron mediator.

FIG. 27A shows an exemplary embodiment of the present invention, showinga biochemical response of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), maltose (shownas a triangle), an xylose (shown as a rectangle). FIG. 27B shows thebiochemical response of FAD-GDHα (N177S, N215S, F353L, F406L) to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beencalculated. FAD-GDHα (N177S, N215S, F353L, F406L) activity wasdetermined by monitoring a decrease of dichlorophenolindophenol (DCIP)signal at OD₆₀₀ (Epoch Microplate Spectrophotometer, Biotek).

FIGS. 28A and 28B, show an electrochemical response to varioussubstrates. In an exemplary embodiment, FIG. 28A shows electrochemicaldata representing the current response of the biosensor to variousglucose concentrations (shown as a rhombus), maltose concentrations(shown as a triangle) and xylose (shown as a rectangle) concentrations.FIG. 28B shows a biosensor response to glucose and the non-linear fitthrough which apparent K_(m) (k) and V_(max) have been calculated. In anexemplary embodiment, wild type FAD-GDHα was immobilized to a carbonelectrode via an electropolymerization method as described previously.R² represents a linear fit of the glucose data. FIG. 28B showsK_(mapp)=0.85 mM and I_(max)=1218.5 nA.

FIG. 29A shows biochemical responses of wild type FAD-GDHα (w.t.),varying concentrations of glucose (rhombus), maltose (triangle) andxylose (rectangle).

FIG. 29B shows a biochemical response of wild type FAD-GDHα to glucoseand the non-linear fit through which Km (k) and V_(max) have beencalculated. The enzyme activity of wild type FAD-GDHα was determined bymonitoring the decrease of Dichlorophenolindophenol (DCIP) signal atOD₆₀₀.

FIG. 30 is a table showing the results obtained from the biochemical andelectrochemical experiments characterizing FAD-GDHα (N177S, N215S,F353L, F406L) where: K_(m) is the Michaelis constant, K_(cat) is theenzyme's catalytic constant, K_(cat)/K_(m) is the catalytic efficiency,glucose/xylose is the ratio of K_(catglu)/K_(catxyl), BEC linearity isthe R² of the linear fit of the bioelectrochemical experiment, I₂₀ isthe current flux (nA/cm²) measured when 20 mM of glucose was tested,Xylose selectivity is the ratio of I_(20glu)/I_(20xyl), Maltoseselectivity is the ratio of I_(20glu)/I_(20mal), pos1-4, the mutatedamino acid number.

FIG. 31 shows sequence data of several mutated FAD-GDH proteinsaccording to some embodiments of the present invention.

FIG. 32 shows a table of electrochemistry data of the embodiments of thecomposition of the present invention. Mutations in position 406 provideimproved linearity over the entire range of physiological range:F406-S/C/T/V/Y/N/P/L/G/A/I/D/E.

FIG. 33 shows some aspects of some embodiments of the present invention.Mutations in position 406 that provide improved selectivity of glucose:F406-S/C/T/M/V/Y/N/P/L/G/Q/A/I/D/H/E. F406W provides an example of asubstitution that reduces the enzyme selectivity towards glucose.

FIG. 34A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention. FIG. 34B shows the non-linear fit (red line) of the datarepresented in FIG. 34A. V_(max) refers to the maximum current flux, Krefers to the apparent K_(m) value extracted from the Michaelis mentenequation.

FIG. 35A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, via direct electron transfer. FIG. 35B shows the non-linearfit (red line) of the data represented in FIG. 35A. V_(max) refers tothe maximum current flux, K refers to the apparent K_(m) value extractedfrom the Michaelis menten equation.

FIG. 36A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, via direct electron transfer. FIG. 36B shows the non-linearfit (red line) of the data represented in FIG. 36A. V_(max) refers tothe maximum current flux, K refers to the apparent K_(m) value extractedfrom the Michaelis menten equation.

FIG. 37A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, via direct electron transfer. FIG. 37B shows the non-linearfit (red line) of the data represented in FIG. 37A. V_(max) refers tothe maximum current flux, K refers to the apparent K_(m) value extractedfrom the Michaelis menten equation.

FIGS. 38A and 38B shows an exemplary embodiment of the presentinvention, showing electrochemistry data of FAD-GDHα (N177S, N215S,F353L, F406L), using an electrode according to some embodiments of thepresent invention configured to measure glucose levels continuously, for20 hrs (FIG. 38A), or 64 hr (FIG. 38B).

FIG. 39 shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L), using anelectrode according to some embodiments of the present inventionconfigured to measure glucose levels continuously, for 20 hrs.

FIG. 40 shows a table of electrochemistry data of the embodiments of theelectrodes of the present invention.

FIGS. 41A to 41C shows a sequence alignment for multiple differentFAD-GDHα proteins. For the consensus sequence, uppercase indicatesidentity, lowercase indicates consensus level of greaters than 0.5, ! isany one of I or V, $ is an one of L or M, % is any one of F or Y, # isany one of NDQEBZ.

Among those benefits and improvements that have been disclosed, otherobjects and advantages of this invention will become apparent from thefollowing description taken in conjunction with the accompanyingfigures. Detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely illustrative of the invention that may be embodied in variousforms. In addition, each of the examples given in connection with thevarious embodiments of the invention which are intended to beillustrative, and not restrictive.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention will be further explained with reference to theattached drawings, wherein like structures are referred to by likenumerals throughout the several views. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the present invention. Further, somefeatures may be exaggerated to show details of particular components.

The figures constitute a part of this specification and includeillustrative embodiments of the present invention and illustrate variousobjects and features thereof. Further, the figures are not necessarilyto scale, some features may be exaggerated to show details of particularcomponents. In addition, any measurements, specifications and the likeshown in the figures are intended to be illustrative, and notrestrictive. Therefore, specific structural and functional detailsdisclosed herein are not to be interpreted as limiting, but merely as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrases “in one embodiment” and “in someembodiments” as used herein do not necessarily refer to the sameembodiment(s), though it may. Furthermore, the phrases “in anotherembodiment” and “in some other embodiments” as used herein do notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, throughout the specification, the meaning of “a,” “an,” and“the” include plural references. The meaning of “in” includes “in” and“on.”

As used herein, “linearity” refers to the R² value of a linear fit whichis calculated for the plot of the relation between substrateconcentration and the measured current. In some embodiments, goodlinearity is considered as an R² value equal or above 0.85 across theentire range of physiological glucose level (0-600 mg/dL). In someembodiments, good linearity is considered to be an R² value that isequal or above 0.9 across the entire range of physiological glucoselevel. In some embodiments, good linearity is considered as an R² valueequal or above 0.95 across the entire range of physiological glucoselevel. Glucose sensing devices transform the measured current to glucoselevel using a linear based algorithm. Thus the linear range of theglucose sensing enzyme improves the accuracy of measurement of the bloodglucose concentration.

As used herein, “direct electron transfer” means the ability of anenzyme to conduct electrons from its enzymatic core to an electrode,during catalysis, generating a detectable current.

The flavoprotein Glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is quiterecently utilized as a glucose sensing enzyme in glucose test strips.The FAD-GDH catalyzes the oxidation of glucose through the use ofvarious electron acceptors (such as Dichlorophenolindophenol).

To date FAD-GDH has been isolated from gram negative bacteria(Burkholderia cepacia), fungi (Aspergillus sp., A. oryzae, A. niger, A.terreus) and from insects (Drosophila melanogaster, Anopheles gambiae,Apis mellifera, Tribolium castaneum).

The protein is composed of three subunits: a catalytic subunit harboringFAD at its redox center (alpha, 67 kDa), a multiheme electron-transfersubunit (beta, 43 kDa) and a chaperon subunit (gamma, 20 kDa). The alphasubunit can be recombinantly expressed and purified in E. Coliindependently of the other subunits, as well as with the beta and thegamma subunits while maintaining catalytic activity in either cases.FAD-GDH is an enzyme that catalyses the oxidation of glucose in thepresence of an electron acceptor, such as 2,6-dichlorophenolindophenolor potassium ferricyanide. FAD-GDH can be used in analyte detectionassays. FAD-GDH is comprised of multiple subunits, including thecatalytic subunit alpha.

In some embodiments the FAD-GDH of the present invention, including allmutants described in the present invention, can be expressed with a beta(β) subunit (a cytochrome domain) in tandem or on a different plasmid,expressed and purified to generate a protein which can deliver improvedelectron transfer to various biosensor applications. In someembodiments, the FAD-GDH of the present invention can be expressed andpurified without a beta (β) subunit, as further detailed below.

In some embodiments, analyte detection assays, e.g., glucose detectionassays, can be based on the production of hydrogen peroxide and thesubsequent detection thereof. For example, glucose is quantitated usingassays by first oxidizing glucose with glucose oxidase to producegluconic acid and hydrogen peroxide. The resultant hydrogen peroxide, inconjunction with a peroxidase, causes the conversion of one or moreorganic substrates, i.e. an indicator, into a chromogenic product, whichproduct is then detected and related to the glucose concentration in theinitial sample.

In the present invention, the inventors have mutated the alpha subunitand co-expressed it with a native gamma subunit. The artificiallymutated FAD-GDH alpha (FAD-GDHa) subunit of the present invention can beco-expressed with either a native or a mutated gamma subunit, betasubunit, or both. For example, in some embodiments, the enzymeelectrode, configured to measure the amount of glucose in aphysiological fluid, comprising the mutated FAD-GDHα protein accordingto some embodiments of the present invention immobilized onto theelectrode further comprises at least one subunit selected from the groupconsisting of: wild-type FAD-GDHβ subunit, and a wild-type FAD-GDHγsubunit.

In some embodiments, the present invention is a mutated FAD-GDHαprotein, wherein the mutated FAD-GDHα protein is mutated from awild-type first species to contain at least one point mutation, whereinthe mutated FAD-GDHα protein comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)=₁G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X)_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of X, S, C, T, M, V,        Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or        V, then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is selected from the group consisting of S and X; and    -   d) X⁵ is selected from the group consisting of S and X.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is selected from the group consisting of S and X; and    -   d) X⁵ is selected from the group consisting of S and X.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In some embodiments,

-   -   a) X¹ is X;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is S; and    -   d) X⁵ is X.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is X; and    -   d) X⁵ is S.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is X;    -   c) X⁴ is S; and    -   d) X⁵ is S.

In some embodiments,

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is S; and    -   d) X⁵ is S.

In some embodiments, the amino acid sequence of the mutated FAD-GDHαprotein comprises the amino acid sequence set forth in any one of SEQ IDNOS: 9-29, SEQ ID NOS: 46-104, or SEQ ID NOS: 105-129.

In some embodiments, a biochemical activity is increased at least 10%compared to a non-mutated FAD-GDHα protein from the wild type firstspecies.

In some embodiments, a selectivity for glucose is increased at least 10%compared to a non-mutated FAD-GDHα protein from the wild type firstspecies.

In some embodiments, a linearity of current as a function of glucoseconcentration is increased at least 10% compared to a non-mutatedFAD-GDHα protein from the wild type first species.

In some embodiments, the FAD-GDHα protein has at least one pointmutation, wherein the point mutation can be at any one of the amino acidresidues selected from the group consisting of: amino acid 177, aminoacid 215, amino acid 353, and amino acid 406.

In some embodiments, the FAD-GDHα protein has a point mutation at aminoacid 406, and at least one additional point mutation, wherein the atleast one additional point mutation can be at any one of the amino acidresidues selected from the group consisting of: amino acid 177, aminoacid 215, and amino acid 353.

In some embodiments, the FAD-GDHα protein has point mutations at aminoacids 177, 215, and 406.

In some embodiments, the FAD-GDHα protein has point mutations at aminoacids 215, 353, and 406.

In some embodiments, the FAD-GDHα protein has point mutations at aminoacids 177, 215, 353, and 406.

A person of ordinary skill in the art would understand that to create aprotein with Flavin Adenine Dinucleotide—Glucose Dehydrogenase alphasubunit (FAD-GDHa) activity according to the instant invention, onewould select amino acid residues for such a protein based on the aminoacid sequence of a naturally-occurring FAD-GDHα protein. The amino acidresidues specified in the above sequences (i.e. those residues notidentified by “X”) represent residues that are conserved among FAD-GDHαproteins. Such residues serve as a reference point to better specify forthe person of ordinary skill in the art the location of amino acidresidues in naturally-occurring FAD-GDHα (i.e. those identified hereinas “X¹” and “X²”) which can be mutated to create a nonnaturally-occurring FAD-GDHα protein with improved properties.

In some embodiments, the FAD-GDHα proteins of the instant inventionincludes at least one mutation (e.g., a point mutation) in the aminoacid sequence, e.g., SEQ ID NOs: 3-8, e.g., as shown in Tables 2 to 6.In some embodiments, the mutation is located at methionine 43 ofFAD-GDHa. In some embodiments, the mutation is located at isoleucine 346of FAD-GDHa. In some embodiments, the mutation is located at serine 420of FAD-GDHa. In some embodiments, the mutation is located at serine 365of FAD-GDHa. In some embodiments, the mutation is located at glycine 208of FAD-GDHa. In some embodiments, the mutation is located at threonine521 of FAD-GDHa. In some embodiments, the mutation is located at valine306 of FAD-GDHa. In some embodiments, the mutation is located atglutamine 412 of FAD-GDHa. In some embodiments, the mutation is locatedat arginine 416 of FAD-GDHa. In some embodiments, the mutation islocated at asparagine 215 of FAD-GDHa. In some embodiments, the mutationis located at alanine 487 of FAD-GDHa. In some embodiments, the mutationis located at asparagine 116 of FAD-GDHa. In some embodiments, themutation is located at asparagine 177 of FAD-GDHa. In some embodiments,the mutation is located at methionine 219 of FAD-GDHa. In someembodiments, the mutation is located at aspartic acid 440 of FAD-GDHa.In some embodiments, the mutation is located at serine 330 of FAD-GDHa.In some embodiments, the mutation is located at proline 257 of FAD-GDHa.In some embodiments, the mutation is located at asparagine 474 ofFAD-GDHa. In some embodiments, the mutation is located at threonine 521of FAD-GDHa. In some embodiments, the mutation is located at serine 420of FAD-GDHa. In some embodiments, the mutation is located at serine 365of FAD-GDHa. In some embodiments, the mutation is located at isoleucine261 of FAD-GDHa. In some embodiments, the mutation is located atthreonine 521 of FAD-GDHa. In some embodiments, the mutation is locatedat proline 173 of FAD-GDHa. In some embodiments, the mutation is locatedat methionine 219 of FAD-GDHa. In some embodiments, the mutation islocated at aspartic acid 301 of FAD-GDHa. In some embodiments, themutation is located at phenylalanine 353 of FAD-GDHa. In someembodiments, the mutation is located threonine 521 of FAD-GDHa. In someembodiments, the mutation is located phenylalanine 406 of FAD-GDHa. Insome embodiments, FAD-GDHα has at least one mutation (e.g., 1 mutation,2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12mutations, 13 mutations, 14 mutations, 15 mutations, etc.). In someembodiments, FAD-GDHα has at least two mutations (e.g., 2 mutations, 3mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13mutations, 14 mutations, 15 mutations, etc.). In some embodiments, themutations are point mutations. In some embodiments, asparagine 475 ofFAD-GDHα is not mutated.

Artificially mutated FAD-GDHα protein is meant to refer to a FAD-GDHαprotein which has at least one amino acid difference from anaturally-occurring FAD-GDHα protein. In some embodiments, the presentinvention is a protein, including: an artificially mutated FAD-GDHαprotein including at least one mutation, where the at least one mutation(e.g., but not limited to, 1 mutation, 2 mutations, 3 mutations, 4mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14mutations, 15 mutations, etc.) is at position 406 of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the at least one mutation isselected from the group including: F406S, F406C, F406T, F406M, F406V,F406Y, F406N, F406P, F406L, F406G, F406Q, F406A, F406I, F406D, F406W,F406H, and F406E. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits at least a 10% increase (e.g., but not limited to, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in biochemicalactivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 10%-400%increase in biochemical activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 10%-350% increase in biochemical activity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 10%-300% increase in biochemicalactivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 10%-250%increase in biochemical activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 10%-200% increase in biochemical activity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 10%-150% increase in biochemicalactivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 10%-100%increase in biochemical activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 10%-50% increase in biochemical activity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-400% increase in biochemical activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 100%-400% increase in biochemicalactivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150%-400%increase in biochemical activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 200%-400% increase in biochemical activity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 250%-400% increase inbiochemical activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 300%-400% increase in biochemical activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 350%-400% increase in biochemical activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-350% increase in biochemical activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 100%-300% increase in biochemicalactivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150%-250%increase in biochemical activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 200%-250% increase in biochemical activity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 150%-200% increase inbiochemical activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 40% increase (e.g., but not limited to, 40%, 50%, 60%, 70%,80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 40% and 400% increase in selectivity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 100% and 400% increase inselectivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150% and400% increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 200% and 400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 250% and 400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300% and 400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350% and 400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 40% and 350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 40% and 300% increase in selectivity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 40% and 250% increase inselectivity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 40% and200% increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 40% and 150% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 40% and 100% increase in selectivity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 100% and 350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150% and 300% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 200% and 250%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 500% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 50% and 500% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 100% and500% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150% and 500% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially FAD-GDHα proteinexhibits between a 200% and 500% increase in linearity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250% and 500% increase in linearitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300% and 500%increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350% and 500% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 400% and 500% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 450% and 500% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 450% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 400% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 20% and 350% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and300% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 250% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 200% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 20% and 150% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and100% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 50% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 50% and 450% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 100% and 400% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150% and350% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 200% and 300% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200% and 250% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 250% and 300% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

The present invention is a protein, including: an artificially mutatedFAD-GDHα protein including at least one mutation (e.g., but not limitedto, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations,etc.), wherein the at least one mutation is at position 474 of SEQ IDNO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutationis selected from the group consisting of: N474H, N474L, N474S and N474V.In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 150%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 200%-400% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 250%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 300%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 20%-300% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-250% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-200% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-100%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20%-50% increase in activity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 100%-350% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150%-300% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 200%-250% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350%-400% increase in selectivity compared to aFAD-GDHαwild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-200%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-150% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-100% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-50% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 100%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 150%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 200%-250%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-200% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

The present invention is a protein, including: an artificially mutatedFAD-GDHα protein including at least one mutation (e.g., but not limitedto, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations,etc.), wherein the at least one mutation is at position 177 of SEQ IDNO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutationis N177S.

In some embodiments, the mutation is located at asparagine 177 ofFAD-GDHα of B. cepacia. In some embodiments, the mutation is located atasparagine 177 of FAD-GDHα and the asparagine is mutated to a polaramino acid, e.g., but not limited to, serine, threonine, cysteine,tyrosine, arginine, and glutamine. In some embodiments, the mutation islocated at asparagine 177 of FAD-GDHα and the asparagine is mutated to apolar amino acid, e.g., but not limited to, serine, threonine, andcysteine. In some embodiments, the mutation is located at asparagine 177of FAD-GDHα and the asparagine is mutated to a polar amino acid, e.g.,serine. In some embodiments, the mutation is located at asparagine 177of FAD-GDHα and the asparagine is mutated to a basic amino acid, e.g.,but not limited to, histidine and lysine. In some embodiments, themutation is located at asparagine 177 of FAD-GDHα and the asparagine ismutated to an acidic amino acid, e.g., but not limited to, aspartic acidand glutamic acid. In some embodiments, the mutation is located atasparagine 177 of FAD-GDHα and the asparagine is mutated to a neutralamino acid, e.g., but not limited to, tryptophan, phenylalanine,glycine, alanine, valine, isoleucine, and leucine.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 150%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 200%-400% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 250%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 300%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 20%-300% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-250% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-200% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-100%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20%-50% increase in activity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 100%-350% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150%-300% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 200%-250% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-200%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-150% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-100% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-50% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 100%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 150%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 200%-250%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-200% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 500% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 50% and 500% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 100% and500% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150% and 500% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially FAD-GDHα proteinexhibits between a 200% and 500% increase in linearity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250% and 500% increase in linearitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300% and 500%increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350% and 500% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 400% and 500% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 450% and 500% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 450% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 400% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 20% and 350% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and300% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 250% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 200% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 20% and 150% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and100% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 50% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 50% and 450% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 100% and 400% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150% and350% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 200% and 300% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200% and 250% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 250% and 300% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20% and 500% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 50% and500% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100% and 500%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 150% and 500% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially FAD-GDHα protein exhibitsbetween a 200% and 500% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 250% and 500% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 300% and500% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350% and 500%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 400% and 500% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 450% and 500% increase in detectable current viadirect electron transport compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 450% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20% and 400% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and350% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20% and 300%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20% and 250% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 20% and 200% increase in detectable current viadirect electron transport compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 150% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20% and 100% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and 50%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50% and 450% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 100% and 400% increase in detectable current viadirect electron transport compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150% and 350% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 200% and 300% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 200% and250% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 250% and 300%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39.

The present invention is a protein, including: an artificially mutatedFAD-GDHα protein including at least one mutation (e.g., but not limitedto, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations,etc.), wherein the at least one mutation is at position 353 of SEQ IDNO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutationis F353L.

In some embodiments, the mutation is located at phenylalanine 353 ofFAD-GDHα of B. cepacia. In some embodiments, the mutation is located atphenylalanine 353 of FAD-GDHα and the asparagine is mutated to a neutralamino acid, e.g., but not limited to, tryptophan, glycine, alanine,valine, isoleucine, and leucine. In some embodiments, the mutation islocated at phenylalanine 353 of FAD-GDHα and the phenylalanine ismutated to a neutral amino acid, e.g., but not limited to, glycine,alanine, valine, isoleucine, and leucine. In some embodiments, themutation is located at phenylalanine 353 of FAD-GDHα and thephenylalanine is mutated to a neutral amino acid, e.g., but not limitedto, valine, isoleucine, and leucine. In some embodiments, the mutationis located at phenylalanine 353 of FAD-GDHα and the phenylalanine ismutated to a neutral amino acid, e.g., but not limited to, leucine. Insome embodiments, the mutation is located at phenylalanine 353 ofFAD-GDHα and the phenylalanine is mutated to a basic amino acid, e.g.,but not limited to, histidine and lysine. In some embodiments, themutation is located at phenylalanine 353 of FAD-GDHα and thephenylalanine is mutated to a polar amino acid, e.g., but not limitedto, serine, threonine, cysteine, tyrosine, arginine, asparagine, andglutamine. In some embodiments, the mutation is located at phenylalanine353 of FAD-GDHα and the phenylalanine is mutated to an acidic aminoacid, e.g., but not limited to, aspartic acid and glutamic acid.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 150%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 200%-400% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 250%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 300%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 20%-300% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-250% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-200% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-100%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20%-50% increase in activity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 100%-350% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150%-300% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 200%-250% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-200%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-150% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-100% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-50% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 100%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 150%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 200%-250%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-200% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 500% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 50% and 500% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 100% and500% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150% and 500% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially FAD-GDHα proteinexhibits between a 200% and 500% increase in linearity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250% and 500% increase in linearitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300% and 500%increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350% and 500% increase in linearity compared to aFAD-GDHαwild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 400% and 500% increase in linearitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 450% and 500%increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 450% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 400% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 20% and 350% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and300% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 250% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 200% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHαprotein exhibits between a 20% and 150% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and100% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 50% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 50% and 450% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 100% and 400% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150% and350% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 200% and 300% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200% and 250% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 250% and 300% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20% and 500% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 50% and500% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100% and 500%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 150% and 500% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially FAD-GDHα protein exhibitsbetween a 200% and 500% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 250% and 500% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 300% and500% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350% and 500%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 400% and 500% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 450% and 500% increase in detectable current viadirect electron transport compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 450% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20% and 400% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and350% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20% and 300%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20% and 250% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 20% and 200% increase in detectable current viadirect electron transport compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 150% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20% and 100% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and 50%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50% and 450% increase in detectablecurrent via direct electron transport compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 100% and 400% increase in detectable current viadirect electron transport compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150% and 350% increase in detectable current via directelectron transport compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 200% and 300% increase in detectable current via direct electrontransport compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 200% and250% increase in detectable current via direct electron transportcompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 250% and 300%increase in detectable current via direct electron transport compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39.

The present invention is a protein, including: an artificially mutatedFAD-GDHα protein including at least one mutation (e.g., but not limitedto, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations,etc.), wherein the at least one mutation is at position 215 of SEQ IDNO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutationis selected from the group consisting of: N215G, N215H, N215T, N215D,N215Y, and N215S.

In some embodiments, the mutation is located at asparagine 215 ofFAD-GDHα of B. cepacia. In some embodiments, the mutation is located atasparagine 215 of FAD-GDHα and the asparagine is mutated to a polaramino acid, e.g., but not limited to, serine, threonine, cysteine,tyrosine, arginine, and glutamine. In some embodiments, the mutation islocated at asparagine 215 of FAD-GDHα and the asparagine is mutated to apolar amino acid, e.g., but not limited to, serine, threonine, andcysteine. In some embodiments, the mutation is located at asparagine 215of FAD-GDHα and the asparagine is mutated to a polar amino acid, e.g.,serine. In some embodiments, the mutation is located at asparagine 215of FAD-GDHα and the asparagine is mutated to a basic amino acid, e.g.,but not limited to, histidine and lysine. In some embodiments, themutation is located at asparagine 215 of FAD-GDHα and the asparagine ismutated to an acidic amino acid, e.g., but not limited to, aspartic acidand glutamic acid. In some embodiments, the mutation is located atasparagine 215 of FAD-GDHα and the asparagine is mutated to a neutralamino acid, e.g., but not limited to, tryptophan, phenylalanine,glycine, alanine, valine, isoleucine, and leucine.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 150%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 200%-400% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 250%-400% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 300%-400% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350%-400%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 20%-300% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-250% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-200% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-100%increase in activity compared to a FAD-GDHα wild-type protein comprisingan amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In someembodiments, the artificially mutated FAD-GDHα protein exhibits betweena 20%-50% increase in activity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-400% increase in activity compared to a FAD-GDHα wild-typeprotein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO:39. In some embodiments, the artificially mutated FAD-GDHα proteinexhibits between a 100%-350% increase in activity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150%-300% increase in activity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 200%-250% increase in activitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 50%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 100%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200%-400% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250%-400% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300%-400%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350%-400% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-200%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-150% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-100% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-50% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-350% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 100%-300% increase in selectivity compared toa FAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 150%-250% increase in selectivitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 200%-250%increase in selectivity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150%-200% increase in selectivity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 500% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 50% and 500% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 100% and500% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 150% and 500% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially FAD-GDHα proteinexhibits between a 200% and 500% increase in linearity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 250% and 500% increase in linearitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 300% and 500%increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 350% and 500% increase in linearity compared to aFAD-GDHαwild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 400% and 500% increase in linearitycompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 450% and 500%increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 450% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 400% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 20% and 350% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and300% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 250% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20% and 200% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHαprotein exhibits between a 20% and 150% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 20% and100% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20% and 50% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 50% and 450% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 100% and 400% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 150% and350% increase in linearity compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 200% and 300% increase in linearity compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 200% and 250% increase in linearity comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence of SEQID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificiallymutated FAD-GDHα protein exhibits between a 250% and 300% increase inlinearity compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsat least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, etc.) in current response compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutatedFAD-GDHα protein exhibits between a 20%-400% increase in currentresponse compared to a FAD-GDHα wild-type protein comprising an aminoacid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments,the artificially mutated FAD-GDHα protein exhibits between a 50%-400%increase in current response compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 100%-400% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150%-400% increase in current responsecompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 200%-400%increase in current response compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 250%-400% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 300%-400% increase in current responsecompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 350%-400%increase in current response compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-350% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-300% increase in current responsecompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-250%increase in current response compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-200% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 20%-150% increase in current responsecompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 20%-100%increase in current response compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 20%-50% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39.

In some embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 50%-350% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 100%-300% increase in current responsecompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, theartificially mutated FAD-GDHα protein exhibits between a 150%-250%increase in current response compared to a FAD-GDHα wild-type proteincomprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, the artificially mutated FAD-GDHα protein exhibitsbetween a 200%-250% increase in current response compared to a FAD-GDHαwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 orSEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHαprotein exhibits between a 150%-200% increase in current responsecompared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the at least one mutation is selected from thegroup including: F406S, F406C, F406T, F406M, F406V, F406Y, F406N, F406P,F406L, F406G, F406Q, F406A, F406I, F406D, F406W, F406H, and F406Eresults in at least a 20% increase (e.g., but not limited to, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to aFAD-GDHα wild-type protein comprising an amino acid sequence of SEQ IDNO: 38 or SEQ ID NO: 39, but a decrease in the current response,compared to a FAD-GDHα wild-type protein comprising an amino acidsequence of SEQ ID NO: 38 or SEQ ID NO: 39.

In some embodiments, the at least one mutation is selected from thegroup consisting of: N215G, N215H, N215T, N215D, N215Y, and N215Sresults in at least a 20% increase (e.g., but not limited to, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in current response comparedto a FAD-GDHα wild-type protein comprising an amino acid sequence havingat least one mutation is selected from the group including: F406S,F406C, F406T, F406M, F406V, F406Y, F406N, F406P, F406L, F406G, F406Q,F406A, F406I, F406D, F406W, F406H, and F406E.

In some embodiments, the protein of the present invention includes atleast one mutation at position 406 of SEQ ID NO: 38 or SEQ ID NO: 39,where a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO:39 is replaced with any amino acid other than K or R.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 95% sequence identity (e.g., but notlimited to, 95%, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39.In some embodiments, a phenylalanine at the position 406 of SEQ ID NO:38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 96% sequence identity (e.g., but notlimited to, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 97% sequence identity (e.g., but notlimited to, 97%, 97.5%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. Insome embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 98% sequence identity (e.g., but notlimited to, 98%, 98.1%, 98.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39.In some embodiments, a phenylalanine at the position 406 of SEQ ID NO:38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 99% sequence identity (e.g., but notlimited to, 99%, 99.1%, 99.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39.In some embodiments, a phenylalanine at the position 406 of SEQ ID NO:38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 95% sequence identity (e.g., but notlimited to, 95%, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39,where the asparagine residue at position 474 is substituted with valine,histidine, leucine, or serine.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 96% sequence identity (e.g., but notlimited to, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39,where the asparagine residue at position 474 is substituted with valine,histidine, leucine, or serine.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 97% sequence identity (e.g., but notlimited to, 97%, 97.5%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39,where the asparagine residue at position 474 is substituted with valine,histidine, leucine, or serine.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 98% sequence identity (e.g., but notlimited to, 98%, 98.1%, 98.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39,where the asparagine residue at position 474 is substituted with valine,histidine, leucine, or serine.

In some embodiments, a mutated FAD-GDHα protein, having at least oneamino acid mutation, has at least 99% sequence identity (e.g., but notlimited to, 99%, 99.1%, 99.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39,where the asparagine residue at position 474 is substituted with valine,histidine, leucine, or serine.

In some embodiments, the present invention is a FAD-GDHα protein havingat least 95% sequence identity to any of SEQ IDS: 9-29, 40-66, or86-104. In some embodiments, the present invention is a FAD-GDHα proteinhaving at least 96% sequence identity to any of SEQ IDS: 9-29, 40-66, or86-104. In some embodiments, the present invention is a FAD-GDHα proteinhaving at least 97% sequence identity to any of SEQ IDS: 9-29, 40-66, or86-104. In some embodiments, the present invention is a FAD-GDHα proteinhaving at least 98% sequence identity to any of SEQ IDS: 9-29, 40-66, or86-104. In some embodiments, the present invention is a FAD-GDHα proteinhaving at least 99% sequence identity to any of SEQ IDS: 9-29, 40-66, or86-104.

In some embodiments, the present invention is a FAD-GDHα protein of anyone of SEQ IDs: 9-29, 40-66, or 86-104 having between 1-5 amino acidmutations. In some embodiments, the present invention is a FAD-GDHαprotein of any one of SEQ IDs: 9-29, 40-66, or 86-104 having between1-10 amino acid mutations. In some embodiments, the present invention isa FAD-GDHα protein of any one of SEQ IDs: 9-29, 40-66, or 86-104 havingbetween 1-15 amino acid mutations. Proteins of the instant invention areunderstood to comprise the full length of the amino acid sequencedescribed in such SEQ ID NOs and not a subset.

In some embodiments, the present invention is a FAD-GDHα protein (SEQ IDNO: 38 or SEQ ID NO: 39) wherein the FAD-GDHα protein includes between1-15 amino acid substitutions, where at least one amino acidsubstitution includes a substitution of the phenylalanine at position406 to another other amino acid other than lysine or arginine.

In some embodiments, the present invention is a FAD-GDHα protein(SEQID:1) wherein the FAD-GDHα protein includes between 1-15 amino acidsubstitutions, where at least one amino acid substitution includes asubstitution of the arginine at position 474 to a histidine, leucine,serine, or valine.

In some embodiments, the present invention is a method for measuring theglucose level of a subject, the method including: contacting a bodyfluid obtained from the subject with a mutant FAD-GDHα protein,measuring the current generated by the mutant FAD-GDHα protein,calculating the measured current to a glucose level, or any combinationthereof. In some embodiments, the mutant FAD-GDHα protein includesbetween 1-15 amino acid substitutions including, e.g., but not limitedto, a position of 406, 215, or 474 of SEQ ID NO: 38 or SEQ ID NO: 39.

Amino acids of the present invention include, but are not limited to the20 commonly occurring amino acids. Also included are naturally occurringand synthetic derivatives, for example, selenocysteine. Amino acidsfurther include amino acid analogs. An amino acid “analog” is achemically related form of the amino acid having a differentconfiguration, for example, an isomer, or a D-configuration rather thanan L-configuration, or an organic molecule with the approximate size andshape of the amino acid, or an amino acid with modification to the atomsthat are involved in the peptide bond, so as to be protease resistantwhen polymerized in a polypeptide.

The phrases “amino acid” and “amino acid sequence” as defined here andin the claims can include one or more components which are amino acidderivatives and/or amino acid analogs comprising part or the entirety ofthe residues for any one or more of the 20 naturally occurring aminoacids indicated by that sequence. For example, in an amino acid sequencehaving one or more tyrosine residues, a portion of one or more of thoseresidues can be substituted with homotyrosine.

The one letter and three letter amino acid codes (and the amino acidthat each represents) are as follows: A means ala (alanine); C means cys(cysteine); D means asp (aspartic acid); E means glu (glutamic acid); Fmeans phe (phenylalanine); G means gly (glycine); H means his(histidine); I means ile (isoleucine); K means lys (lysine); L means leu(leucine); M means met (methionine); N means asn (asparagine); P meanspro (proline); Q means gln (glutamine); R means arg (arginine); S meansser (serine); T means thr (threonine); V means val (valine); W means trp(tryptophan); and Y means tyr (tyrosine).

Naturally occurring residues are divided into groups based on commonside-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu,ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4)basic: asn, gln, his, lys, arg; (5) residues that influence chainorientation: gly, pro; (6) aromatic: trp, tyr, phe. A person skilled inthe art would understand that certain conservative substitution may bemade in the amino acid sequence of a protein. Conservative substitutionsof interest are shown in Table 1.

TABLE 1 Exemplary and Preferred Amino acid substitutions. OriginalExemplary Preferred Ala (A) val; leu; ile val Arg (R) lys; gln; asn lysAsn (N) gln; his; lys; arg gln Asp (D) Glu glu Cys (C) Ser ser Gln (Q)Asn asn Glu (E) Asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine;ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) Ala ala Ser (S) Thrthr Thr (T) Ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; met; phe; ala; norleucine leu

In some embodiments, the composition of the present invention isdirected to a FAD-GDHα including at least one point mutation attached toan enzyme electrode configured to: i) recognize a presence of a testsubject (for example, glucose); ii) catalyze a redox reaction; and iii)transfer an electron generated from the redox reaction to the enzymeelectrode. For purposes of the present invention, “catalyze” and“catalysis” refers to activity of specialized proteins (natural orrecombinant), called enzymes, which lower the activation energynecessary for a reaction to occur.

In some embodiments, an enzyme electrode is constructed by immobilizinga FAD-GDHα alone or FAD-GDHα in complex with the gamma subunit on anelectrode and/or an electrode layer. In some embodiments, a FAD-GDHα isimmobilized by utilizing an immobilization method. In some embodimentsof the present invention, the immobilization method is selected from thegroup consisting of: i) using a crosslinking reagent; ii)electropylimerization iii) entrapping an enzyme in a macromolecularmatrix; iv) coating the enzyme with a dialysis membrane; and v)immobilizing the enzyme in a polymer, the polymer selected from thegroup consisting of: 1) a photo-crosslinking polymer; 2) an electricconductive polymer; and 3) a redox polymer. In some embodiments,combinations of the immobilization methods can be utilized.

In some embodiments, the present invention is directed to an enzymeelectrode comprising a conductive material and an immobilized or wiredFAD-GDHα. In some embodiments, the enzyme electrode generates adetectable electric current that corresponds to an amount of an analytein a tested sample. In some embodiments, the enzyme electrode is anenzyme electrode including an immobilized enzyme on the surface of theenzyme electrode. In some embodiments of the present invention, theenzyme electrode is selected from the group consisting of a goldelectrode, a platinum electrode, a carbon electrode, and any otherconductive material that is suitable for constructing biosensors. Insome embodiments of the present invention, the enzyme electrode utilizesa reaction specificity of an enzyme for detecting any of a variety ofbiologically active substances in a specific manner.

In some embodiments, the present invention is directed to a sensorincluding an enzyme electrode to function as a working electrode. Insome embodiments of the present invention, the sensor is an assay systemfor electrochemically measuring a concentration of a test substance. Insome embodiments of the present invention, the sensor includes threeelectrodes: i) a working electrode ii) a counter electrode; and iii) areference electrode. In some embodiments of the present invention, thesensor comprises a two-electrode system comprising: i) a workingelectrode and ii) a counter electrode. In some embodiments, the sensorfurther includes a power source. In some embodiments, the power sourceapplies voltage to: i) the working electrode; ii) an ampere meter; andiii) a recorder. In some embodiments, the sensor is a batch type sensor.In some embodiments, the sensor is a flow type sensor. In someembodiments, the sensor is a flow-type sensor, where the flow-typesensor continuously measures blood glucose level. In some embodiments,the two-electrode system comprising the immobilized enzyme is insertedinto a flow of continuously supplied blood sample or dialyzed sample, orinto a blood sample or an interstitial fluid sample. In someembodiments, the three-electrode system comprising the immobilizedenzyme is inserted into a flow of continuously supplied blood sample ordialyzed sample, or into a blood sample or an interstitial fluid sample.

In some embodiments, the present invention also provides a glucosesensor comprising any of the FAD-GDHα proteins of the instant invention.Glucose sensors employing FAD-GDH are known in the art and have beendescribed, for example, in U.S. Pat. No. 8,658,011, which is herebyincorporated by reference. The proteins of the instant invention may beemployed in any biosensor know in the art that is designed to employFAD-GDH.

In some embodiments, the present invention also provides a compositioncomprising the FAD-GDHα of the instant invention and a solid support.Such support may be in the form of a reagent layer or a reagent teststrip. In some embodiments the composition is in a dry or solid state.Reagent layers for glucose sensors are known in the art, and have beendescribed, for example, in U.S. Pat. No. 8,658,011. A person skilled inthe art would understand that any reagent layer known in the artdesigned for use with FAD-GDH can be made to comprise the FAD-GDHαproteins of the instant invention. Reagent test strips are used in thedetermination of the concentration of an analyte, e.g. glucose, in aphysiological sample, e.g. blood. The test strips can include a porousmatrix, one or more members of an analyte oxidation signal producingsystem and at least one hemolyzing agent. In using the subject teststrips for analyte concentration determination, a physiological sampleis applied to the test strip. Next, the appearance of a chromogenicproduct of the signal producing system is detected and related to theconcentration of the analyte in the sample.

Typically, a user inserts a test strip into a meter and lances a fingeror alternate body site to obtain a blood sample. The drawn sample isapplied to the test strip and the meter reads the strip and determinesanalyte concentration, which is then conveyed to the user. For example,the blood glucose meter converts a current generated by the enzymaticreaction in the test strip to a corresponding blood glucose value whichis displayed or otherwise provided to the patient to show the level ofglucose at the time of testing.

In some embodiments, the present invention also provides the FAD-GDHαproteins of the instant invention immobilized to the conductivecomponent of an electrode of a glucose sensor. Electrodes for glucosesensors are known in the art, and have been described, for example, inU.S. Pat. No. 7,497,940, the contents of which are hereby incorporatedby reference. A person skilled in the art would understand that anyelectrode known in the art designed for use with FAD-GDH can be made tocomprise the FAD-GDHα proteins of the instant invention.

In some embodiments, the present invention provides an enzyme electrode,configured to measure the amount of glucose in a physiological fluid,comprising the mutated FAD-GDHα protein according to some embodiments ofthe present invention immobilized onto the electrode, wherein themutated FAD-GDHα protein according to some embodiments of the presentinvention is configured to catalyze glucose in the physiological fluidand produce electrons that are transferred to the electrode therebygenerating an electrical current, wherein the intensity of theelectrical current is indicative of the level of glucose in thephysiological fluid.

As used herein, the term “physiological fluid” refers to blood, saliva,interstitial fluid, cell culture medium, and the like.

In some embodiments, the enzyme electrode is a screen printed electrode

In some embodiments, the enzyme electrode is configured to perform asingle measurement.

An example of an enzyme electrode into which a mutated enzyme accordingto some embodiments of the present invention may be incorporated isdisclosed in U.S. Pat. No. 4,431,507.

Another example of an enzyme electrode into which a mutated enzymeaccording to some embodiments of the present invention may beincorporated is disclosed in U.S. Pat. No. 5,762,770.

Another example of an enzyme electrode into which a mutated enzymeaccording to some embodiments of the present invention may beincorporated is disclosed in U.S. Pat. No. 6,270,637.

In some embodiments, the enzyme electrode is incorporated into a glucosetest strip.

In some embodiments, the mutated FAD-GDHα protein is immobilized on theelectrode in a conductive matrix.

In some embodiments, the conductive matrix is selected from a groupconsisting of carbon paste, graphite paste, graphene oxide, or any otherconductive matrix paste appropriate for use in screen printedelectrodes.

In some embodiments, the conductive matrix is a conductive polymer.

In some embodiments, the conductive polymer is selected from the groupconsisting of: PEDOT, and Polypyrrol.

In some embodiments, the conductive polymer is electro-polymerized onthe electrode together with the mutated FAD-GDHα protein.

In some embodiments, the conductive polymer is chemically polymerized onthe electrode together with the mutated FAD-GDHα protein.

In some embodiments, the mutated FAD-GDHα protein of the presentinvention is immobilized to the electrode by chemical wiring.

In some embodiments, the conductive matrix further comprises an electronmediator.

In one embodiment, the enzyme electrode, configured to measure theamount of glucose in a physiological fluid, comprising the mutatedFAD-GDHα protein according to some embodiments of the present inventionimmobilized onto the electrode further comprises at least one subunitselected from the group consisting of: wild-type FAD-GDHβ subunit, and awild-type FAD-GDHγ subunit.

In some embodiments, the enzyme electrode of the present invention isincorporated into a biosensor configured for subcutaneous continuousglucose measurement, wherein the biosensor is configured to continuallymeasure the amount of glucose in the subject.

In some embodiments, the biosensor is configured to continuously measureglucose for up to 2 weeks. In some embodiments, the biosensor isconfigured to continuously measure glucose for up to one week. In someembodiments, the biosensor is configured to continuously measure glucosefor up to six days. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to five days. In some embodiments,the biosensor is configured to continuously measure glucose for up tofour days. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to three days. In some embodiments,the biosensor is configured to continuously measure glucose for up totwo days. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to one day.

In some embodiments, the biosensor is configured to continuously measureglucose for up to 64 hours. In some embodiments, the biosensor isconfigured to continuously measure glucose for up to 62 hours. In someembodiments, the biosensor is configured to continuously measure glucosefor up to 60 hours. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to 58 hours. In some embodiments,the biosensor is configured to continuously measure glucose for up to 56hours. In some embodiments, the biosensor is configured to continuouslymeasure glucose for up to 54 hours. In some embodiments, the biosensoris configured to continuously measure glucose for up to 52 hours. Insome embodiments, the biosensor is configured to continuously measureglucose for up to 50 hours. In some embodiments, the biosensor isconfigured to continuously measure glucose for up to 48 hours. In someembodiments, the biosensor is configured to continuously measure glucosefor up to 46 hours. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to 44 hours. In some embodiments,the biosensor is configured to continuously measure glucose for up to 42hours. In some embodiments, the biosensor is configured to continuouslymeasure glucose for up to 40 hours. In some embodiments, the biosensoris configured to continuously measure glucose for up to 38 hours. Insome embodiments, the biosensor is configured to continuously measureglucose for up to 36 hours. In some embodiments, the biosensor isconfigured to continuously measure glucose for up to 34 hours. In someembodiments, the biosensor is configured to continuously measure glucosefor up to 32 hours. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to 30 hours. In some embodiments,the biosensor is configured to continuously measure glucose for up to 28hours. In some embodiments, the biosensor is configured to continuouslymeasure glucose for up to 26 hours. In some embodiments, the biosensoris configured to continuously measure glucose for up to 24 hours. Insome embodiments, the biosensor is configured to continuously measureglucose for up to 20 hours. In some embodiments, the biosensor isconfigured to continuously measure glucose for up to 18 hours. In someembodiments, the biosensor is configured to continuously measure glucosefor up to 16 hours. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to 14 hours. In some embodiments,the biosensor is configured to continuously measure glucose for up to 12hours. In some embodiments, the biosensor is configured to continuouslymeasure glucose for up to 10 hours. In some embodiments, the biosensoris configured to continuously measure glucose for up to 8 hours. In someembodiments, the biosensor is configured to continuously measure glucosefor up to 6 hours. In some embodiments, the biosensor is configured tocontinuously measure glucose for up to 4 hours. In some embodiments, thebiosensor is configured to continuously measure glucose for up to 2hours. In some embodiments, the biosensor is configured to continuouslymeasure glucose for up to 1 hour.

An example of a biosensor into which a mutated enzyme according to someembodiments of the present invention may be incorporated is disclosed inU.S. Pat. No. 9,163,273.

Another example of a biosensor into which a mutated enzyme according tosome embodiments of the present invention may be incorporated isdisclosed in U.S. Pat. No. 8,808,532.

Another example of a biosensor into which a mutated enzyme according tosome embodiments of the present invention may be incorporated isdisclosed in U.S. Pat. No. 7,074,307.

In some embodiments, the biosensor comprises the mutated FAD-GDHαprotein according to some embodiments of the present inventionimmobilized onto at least one enzyme electrode, wherein the mutatedFAD-GDHα protein according to some embodiments of the present inventionis configured to catalyze glucose in the subject and generate electronsthat are transferred to the electrode and generate electrical current,wherein the intensity of the electrical current is indicative of thelevel of glucose in the subject.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of X, S, C, T, M, V,        Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or        V, then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is selected from the group consisting of S and X; and    -   d) X⁵ is selected from the group consisting of S and X.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is selected from the group consisting of S and X; and    -   d) X⁵ is selected from the group consisting of S and X.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHPβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)=₇Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is X;    -   b) X³ is selected from the group consisting of G, H, D, Y, S,        and X;    -   c) X⁴ is X; and    -   d) X⁵ is X.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is S; and    -   d) X⁵ is X.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is X; and    -   d) X⁵ is S.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is X;    -   c) X⁴ is S; and    -   d) X⁵ is S.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)X¹(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein:

-   -   a) X¹ is selected from the group consisting of S, C, T, M, V, Y,        N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V,        then X² is D;    -   b) X³ is selected from the group consisting of G, H, D, Y, and        S;    -   c) X⁴ is S; and    -   d) X⁵ is S.

In some embodiments, the mutated FAD-GDHα protein further comprises atleast one subunit selected from the group consisting of: wild-typeFAD-GDHβ subunit, and a wild-type FAD-GDHγ subunit.

In some embodiments, the mutated FAD-GDHα protein that is configured tocatalyze glucose in the subject and generate electrons that aretransferred to the electrode and generate electrical current comprises amutated FAD-GDHα having an amino acid sequence set forth in any one ofSEQ ID NOS: 9-29, SEQ ID NOS: 46-104, or SEQ ID NOS: 105-129.

In some embodiments, the electrode is made from a material selected fromthe group consisting of: carbon fiber, graphite, glassy carbon, gold,silver, copper, platinum, palladium, and metal oxide.

In some embodiments, the metal oxide is indium tin oxide.

In some embodiments, mutant FAD-GDHα, according to some embodiments ofthe present invention is immobilized to a carbon electrode via anelectropolymerization method. Briefly, the 3-array screen printedelectrodes (“SPE”), which contain working, counter and referenceelectrodes, is overlaid with 60 μl of saturated HKCO₃ solution andvoltage applied immediately at 1.2V for 180 seconds (versus Ag/AgCl).The solution is then discarded and washed three times with 60 μl of PBS7.4 supplemented with 0.01M magnesium chloride (“EC buffer”). The SPE isthen overlaid with 60 μl of immobilization solution containing of 0.1Mpyrrole, 0.1M potassium chloride and 1 mg/ml of SZ2 enzyme.Alternatively, the SPE waiss then overlaid with 60 μl of immobilizationsolution containing of 0.01M PEDOT, 0.7% poly styrene sulfonate (70K Mw)and 1 mg/ml of SZ2 enzyme. An external electron mediator (“mediator”)can be added to the immobilization solution at the range of 0.5-10 mM.As used herein, “electron mediator” or “mediator” refers to a chemicalthat transfers electrons from the enzyme (e.g., but not limited to,FAD-GDH) to an anode. Electron mediators can include, but are notlimited to, Quinone/Hydroquinone, Phenanthroline quinone, Quinonediimine/Phenylendiamine, Qunone diimine oxides (Nitrosoanilines),Phenazinium/-radical/Dihydro-phenazine, N-oxides (Resazurin,Bezfuroxan), Hexacyanoferrate III/III, Ferricinium/Ferrocene (carboxylicacid), Ruthenium complexes, Osmium as complexed and/or immobilized stateand any other mediators common in the art. The solution was incubated atroom temperature (RT) for 1 minute prior to applying a set of 10 pulsesat 0.65V for 1 second with 5 second relaxation. The solution was thendiscarded and washed three times with 60 μl of EC buffer for initiationof glucose sensing measurement phase.

In the embodiments utilizing PEDOT, the solution is incubated at roomtemperature (RT) for 1 minute prior to applying 2 cyclic voltammetrycycles (0.2-0.9V 50 mv/sec).

In some embodiments, the electrode is overlaid with immobilizationsolution devoid of any mediator as indicated in the table below. Theseembodiments enable direct electron transfer (“DET”). In someembodiments, the immobilization solution is electropolymerized onto theelectrode as described in the table below. The solution is thendiscarded and washed three times with 60 μl of EC buffer for initiationof glucose sensing measurements phase as detailed in the table below.The electrochemical data was collected using a VMP3 multi-channelpotentiostat (Bio-logic Science instruments SAS, France).

# Composition description Polymerization method Glucose sensing 1 0.25Mpyrrole, 0.25M potassium chloride, Phosphate Buffer Saline 20 CA pulsesof 0.65 V, 0.2 V for 30 minutes pH 7.4 supplemented with 0.01 mM MgCl₂and 0.1% Proclin 150 1 second each with 5 second (Sigma, cat# 49376-u)(“PBSm”) and 0.33 mg/ml of Mut111 enzyme intervals between each pulse 20.01M 3,4-Ethylenedioxythiophene (“EDOT”), 0.1 mM Ply(sodium CV cyclesranging 0.2-0.85 V 0.3 V for 30 minutes 4-styrenesulfonate) (“PSS”),PBSm and 1 mg/ml of Mut111 enzyme with a scanning speed of 0.05 V/Sec

Examples of methods by which mutated enzyme according to someembodiments of the present invention may be incorporated into abiosensor are disclosed in U.S. Pat. No. 9,163,273.

Other examples of methods by which mutated enzyme according to someembodiments of the present invention may be incorporated into abiosensor are disclosed in U.S. Pat. No. 8,808,532.

Although the following exemplary embodiments were conducted withnon-naturally mutated FAD-GDHα derived from B. cepacia, these exemplaryembodiments are non-limiting and can be extended to related bacterialFAD-GDHα, such as, but not limited to, FAD-GDHα derived from B. lata,Burkholderia terrae, Pseudomonas denitrificans, Relsonia pickettii,Yersinia mollretii, Pandoraea sp. and Herbspirilum sropedicae. FIG. 7illustrates the homology of the following species: B. cepacia, H.Seropedicae, B. terrae, P. Dentrificans, Y. mollaretii, R. Pickettii,and Pandoraea. The positions described herein are noted using ellipsoidshapes on FIG. 31.

The following electrochemical exemplary embodiments use conductivepolymers, e.g., polypyrrole. In some embodiments, a conductive polymeris used to generate electrochemical data and includes polypyrrole,polythiophene (e.g., poly(3,4-ethylenedioxythiophene)—“PEDOT”),polyaniline, poly(diphenylamine), polyazine, poly(o-aminophenol, or anycombination thereof.

In some embodiments, the present invention also provides a DNA sequenceencoding any of the proteins of the instant invention. Such DNA sequencemay be expressed according to any technique known in the art. By way ofnon-limiting example, such sequence may be incorporated into a vectorfor expression in a cell. The term “vector” refers to a polynucleotidemolecule capable of carrying and transferring another polynucleotidefragment or sequence to which it has been linked from one location(e.g., a host, a system) to another. The term includes vectors for invivo or in vitro expression systems. As a non-limiting example, vectorscan be in the form of “plasmids” which refer to circular double strandedDNA loops which are typically maintained episomally but may also beintegrated into the host genome. In some embodiments, the presentinvention accordingly provides a host cell comprising the DNA encodingthe protein and a process of producing the protein comprising culturingthe host cell under conditions conducive to the production of theprotein, and recovering the protein.

Exemplary embodiments of the activity of wild type FAD-GDHα (SEQ ID NO:38) is shown in International Application Serial No. PCT/US2015/64125,entitled “Compositions and Methods for Measuring Blood Glucose Levels”,filed on Dec. 4, 2015.

Exemplary embodiments of the activity of mutated GAD-GDHα is inInternational Application Serial No. PCT/US2015/64125, entitled“Compositions and Methods for Measuring Blood Glucose Levels”, filed onDec. 4, 2015.

Exemplary embodiments of the composition of the present invention areshown in FIGS. 1A and 1B. FIG. 1A shows the biochemical response ofFAD-GDHα F406L to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 1B shows the biochemical response of F406L toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406L enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀.

An exemplary embodiment of the composition of the present invention,showing the electrochemical data in connection with FAD-GDHα F406L, isshown in FIG. 2.

Exemplary embodiments of the composition of the present invention areshown in FIGS. 3A-3C. FIG. 3A shows the biochemical response of FAD-GDHαF406A to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 3B shows the biochemical response of F406A to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406A enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 3C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 4A-C. FIG. 4A shows the biochemical response of FAD-GDHαF406C to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 4B shows the iochemical response of F406C to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406C enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 4C shows theelectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 5A-C. FIG. 5A shows the biochemical response of FAD-GDHαF406E to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 5B shows the biochemical response of F406E to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406E enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 5C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 6A-C. FIG. 6A shows the biochemical response of FAD-GDHαF406D to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 6B shows the biochemical response of F406D to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406D enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 6C shows theelectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 7A-C. FIG. 7A shows the biochemical response of FAD-GDHαF406G to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 7B shows the biochemical response of F406G to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406G enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 7C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 8A and 8B. FIG. 8A shows the biochemical response ofFAD-GDHα F406H to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 8B shows the biochemical response of F406H toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406H enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 9A-C. FIG. 9A shows the biochemical response of FAD-GDHαF406I to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 9B shows the biochemical response of F406I to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406I enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 9C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 10A and 10B. FIG. 10A shows the biochemical response ofFAD-GDHα F406M to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 10B shows the biochemical response of F406M toglucose and the non-linear fit through which K_(m) (k) and V_(max) havebeen obtained. F406M enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 11A-C. FIG. 11A shows the biochemical response ofFAD-GDHα F406N to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 11B shows the biochemical response of F406N toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406N enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 11Cshows electrochemical data representing the current response of thebiosensor to various glucose concentrations (shown as a rhombus) and thelinear fit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 12A and 12B. FIG. 12A shows the biochemical response ofFAD-GDHα F406Q to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 12B shows the biochemical response of F406Q toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406Q enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 13A-C. FIG. 13A shows biochemical response of FAD-GDHαF406S to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 13B shows the biochemical response of F406S to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406S enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 13C showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus) and the linearfit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 14A-C. FIG. 14A shows the biochemical response ofFAD-GDHα F406T to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 14B shows biochemical response of F406T toglucose and the non-linear fit through which K_(m) (k) and V_(max) havebeen obtained. F406T enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 14Cshows electrochemical data representing the current response of thebiosensor to various glucose concentrations (shown as a rhombus) and thelinear fit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 15A-B. FIG. 15A shows a biochemical response of FAD-GDHαF406W to varying concentrations of glucose (rhombus), and xylose(rectangle). FIG. 15B shows a biochemical response of F406W to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beenobtained. F406W enzyme activity was determined via monitoring decreaseof Dichlorophenolindophenol (DCIP) signal at OD₆₀₀.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 16A-C. FIG. 16A shows the biochemical response ofFAD-GDHα F406Y to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 16B shows the biochemical response of F406Y toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406Y enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 16Cshows electrochemical data representing the current response of thebiosensor to various glucose concentrations (shown as a rhombus) and thelinear fit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 17A-C. FIG. 17A shows the biochemical response ofFAD-GDHα F406V to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 17B shows the biochemical response of F406V toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406V enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 17Cshows electrochemical data representing the current response of thebiosensor to various glucose concentrations (shown as a rhombus) and thelinear fit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 18A-C. FIG. 18A shows the biochemical response ofFAD-GDHα F406P to varying concentrations of glucose (rhombus), andxylose (rectangle). FIG. 18B shows the biochemical response of F406P toglucose and the non-linear fit through which K_(m)(k) and V_(max) havebeen obtained. F406P enzyme activity was determined via monitoringdecrease of Dichlorophenolindophenol (DCIP) signal at OD₆₀₀. FIG. 18Cshows electrochemical data representing the current response of thebiosensor to various glucose concentrations (shown as a rhombus) and thelinear fit across the data range is represented by the R² value.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 19A-B. FIG. 19A shows electrochemical data representingthe current response of the biosensor comprising of FAD-GDHα N215G tovarious glucose concentrations (shown as a square) and the linear fitacross the data range is represented by the R² value. FIG. 19B shows thenon-linear fit of the data shown in FIG. 19A, from which K_(m) (k) andV_(max) have been calculated.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 20A-B. FIG. 20A shows electrochemical data representingthe current response of the biosensor comprising of FAD-GDHα N215H tovarious glucose concentrations (shown as a square) and the linear fitacross the data range is represented by the R² value. FIG. 20B shows thenon-linear fit of the data shown in FIG. 20A, from which K_(m) (k) andV_(max) have been calculated.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 21A-B. FIG. 21A shows electrochemical data representingthe current response of the biosensor comprising of FAD-GDHα N215T tovarious glucose concentrations (shown as a square) and the linear fitacross the data range is represented by the R² value. FIG. 21B shows thenon-linear fit of the data shown in FIG. 21A, from which K_(m) (k) andV_(max) have been calculated.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 22A-B. FIG. 22A shows electrochemical data representingthe current response of the biosensor comprising of FAD-GDHα N215D tovarious glucose concentrations (shown as a square) and the linear fitacross the data range is represented by the R² value. FIG. 22B shows thenon-linear fit of the data shown in FIG. 22A, from which K_(m) (k) andV_(max) have been calculated.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 23A-B. FIG. 23A shows electrochemical data representingthe current response of the biosensor comprising of FAD-GDHα N215Y tovarious glucose concentrations (shown as a square) and the linear fitacross the data range is represented by the R² value. FIG. 23B shows thenon-linear fit of the data shown in FIG. 23A, from which K_(m) (k) andV_(max) have been calculated.

Exemplary embodiments of the compositions of the present invention areshown in FIGS. 24A-B. FIG. 24A shows electrochemical data representingthe current response of the biosensor comprising of FAD-GDHα N215S tovarious glucose concentrations (shown as a square) and the linear fitacross the data range is represented by the R² value. FIG. 24B shows thenon-linear fit of the data shown in FIG. 24A, from which K_(m) (k) andV_(max) have been calculated.

Exemplary embodiments showing electrochemistry data in connection withthe wild type FAD-GDHα protein are shown in FIG. 25.

Exemplary embodiments are shown in a table listing electrochemistry dataof the composition of the present invention in FIG. 26. Mutations inposition 406 provide improved linearity over the entire range ofphysiological range: F406-S/C/T/V/Y/N/P/L/G/A/I/D/E. Mutations inposition 215 provide improved linearity over the entire range ofphysiological range: N215-G/H/T/D/Y/S.

Exemplary embodiments of the composition of the present invention areshown in FIG. 27. Mutations in position 406 that provide improvedselectivity of glucose: F406-S/C/T/M/V/Y/N/P/L/G/Q/A/I/D/H/E. F406Wprovides an example of a substitution that reduces the enzymeselectivity towards glucose.

FIG. 34A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized withpolypyrrole, and benzoquinone is used as a mediator to obtain a singlemeasurement. FIG. 34B shows the non-linear fit (red line) of the datarepresented in FIG. 34A. V_(max) refers to the maximum current flux, Krefers to the apparent K_(m) value extracted from the Michaelis mentenequation.

FIG. 35A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized withpolypyrrole, to obtain a single measurement via direct electrontransfer. FIG. 35B shows the non-linear fit (red line) of the datarepresented in FIG. 35A. V_(max) refers to the maximum current flux, Krefers to the apparent K_(m) value extracted from the Michaelis mentenequation.

FIG. 36A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized with PEDOT,to obtain a single measurement via direct electron transfer. FIG. 36Bshows the non-linear fit (red line) of the data represented in FIG. 36A.V_(max) refers to the maximum current flux, K refers to the apparentK_(m) value extracted from the Michaelis menten equation.

FIG. 37A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized with grapheneoxide, to obtain a single measurement via direct electron transfer. FIG.37B shows the non-linear fit (red line) of the data represented in FIG.37A. V_(max) refers to the maximum current flux, K refers to theapparent K_(m) value extracted from the Michaelis menten equation.

FIGS. 38A and 38B shows an exemplary embodiment of the presentinvention, showing electrochemistry data of FAD-GDHα (N177S, N215S,F353L, F406L), using an electrode according to some embodiments of thepresent invention configured to measure glucose levels continuously, for20 hrs (FIG. 38A), or 64 hr (FIG. 38B). In this the mutant FAD-GDHprotein is immobilized with polypyrrole, to obtain a single measurementvia direct electron transfer.

FIG. 39 shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L), using anelectrode according to some embodiments of the present inventionconfigured to measure glucose levels continuously, for 20 hrs. In thisembodiment, the mutant FAD-GDH protein is immobilized with PEDOT, toobtain a single measurement via direct electron transfer

FIG. 40 shows a table of electrochemistry data of the embodiments of theelectrodes of the present invention.

Exemplary compositions comprising electrodes according to someembodiments of the present invention are shown in FIGS. 39 and 40.Mutations at positions 177, 215, 353, and 406 provide improvedlinearity, selectivity and detectable current via direct electrontransfer over the entire physiological range. Further, the electrodesaccording to some embodiments of the present invention are capable ofmeasuring glucose for up to 64 hours.

In some embodiments, the protein of the present invention can be linkedto an epitope tag, e.g., but not limited to, a HIS tag, a 6×HIS tag, amaltose binding protein tag, a green fluorescent protein tag, aglutathione-s-transferase tag, a streptavidin tag, etc. The epitope tagcan be separated from the protein by using a linker having, e.g., 1-namino acids in length.

Examples: Mutated FAD-GDH, Alpha Subunit (α), Strain B. cepacia

Examples of mutated FAD-GDHα proteins are disclosed in InternationalApplication Serial No. PCT/US2015/64125, entitled “Compositions andMethods for Measuring Blood Glucose Levels”, filed on Dec. 4, 2015.

Example 1

FAD-GDHα was mutated to include the following point mutation: N215D asshown in SEQ ID NO: 105.

Example 2

FAD-GDHα was mutated to include the following point mutation: N215G asshown in SEQ ID NO: 106.

Example 3

FAD-GDHα was mutated to include the following point mutation: N215H asshown in SEQ ID NO: 107.

Example 4

FAD-GDHα was mutated to include the following point mutation: N215T asshown in SEQ ID NO: 108.

Example 5

FAD-GDHα was mutated to include the following point mutation: N215Y asshown in SEQ ID NO: 109.

Example 6

B. sepacia FAD-GDHα was mutated to include the following point mutation:N177S, N215S, F353L, F406L, as shown in SEQ ID NO: 109.

Example 7

B. lata FAD-GDHα was mutated to include the following point mutation:N177S, N215S, F353L, F406L, as shown in SEQ ID NO: 110.

Example 8

B. lata FAD-GDHα was mutated to include the following point mutation:N215S, as shown in SEQ ID NO: 111.

Example 9

B. lata FAD-GDHα was mutated to include the following point mutation:N215T, as shown in SEQ ID NO: 112.

Example 10

B. cepacia FAD-GDHα was mutated to include the following point mutation:N177S, as shown in SEQ ID NO: 113.

Example 11

B. lata FAD-GDHα was mutated to include the following point mutation:N177S, as shown in SEQ ID NO: 114.

Example 12

B. cepacia FAD-GDHα was mutated to include the following point mutation:F353L, as shown in SEQ ID NO: 115.

Example 13

B. lata FAD-GDHα was mutated to include the following point mutation:F353L, as shown in SEQ ID NO: 116.

Example 14

B. cepacia FAD-GDHα was mutated to include the following point mutation:N177S, F406L, as shown in SEQ ID NO: 117.

Example 15

B. lata FAD-GDHα was mutated to include the following point mutation:N177S, F406L, as shown in SEQ ID NO: 118.

Example 16

B. sepacia FAD-GDHα was mutated to include the following point mutation:F353L, F406L, as shown in SEQ ID NO: 119.

Example 17

B. lata FAD-GDHα was mutated to include the following point mutation:F353L, F406L, as shown in SEQ ID NO: 120.

Example 18

B. sepacia FAD-GDHα was mutated to include the following point mutation:N215S, F406L, as shown in SEQ ID NO: 121.

Example 19

B. lata FAD-GDHα was mutated to include the following point mutation:N215S, F406L, as shown in SEQ ID NO: 122.

Example 20

B. sepacia FAD-GDHα was mutated to include the following point mutation:N177S, F353L, F406L, as shown in SEQ ID NO: 123.

Example 21

B. lata FAD-GDHα was mutated to include the following point mutation:N177S, F353L, F406L, as shown in SEQ ID NO: 124.

Example 22

B. sepacia FAD-GDHα was mutated to include the following point mutation:N177S, N215S, F406L, as shown in SEQ ID NO: 125.

Example 23

B. lata FAD-GDHα was mutated to include the following point mutation:N177S, N215S, F406L, as shown in SEQ ID NO: 126.

Example 24

B. sepacia FAD-GDHα was mutated to include the following point mutation:N215S, F353L, F406L, as shown in SEQ ID NO: 127.

Example 25

B. lata FAD-GDHα was mutated to include the following point mutation:N215S, F353L, F406L, as shown in SEQ ID NO: 128.

The proteins described in Examples 1-25 were generated according to themethods described in International Application Serial No.PCT/US2015/64125, entitled “Compositions and Methods for Measuring BloodGlucose Levels”, filed on Dec. 4, 2015.

TABLE 2 Protein sequences of FAD-GDHγα from B. cepacia (SEQ ID NO: 1,3-29) and B. lata (SEQ ID NO: 2) and DNA sequences of primers used forrandom mutagenesis via error prone PCR. Each protein sequence ends in aremovable 6x-His tag. SEQ ID Sequence SEQ ID NO: 1 Protein sequence ORF2(FADα) - B. cepacia Wild typeMADTDTQKADVVVVGSGVAGATVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPOPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 2 Protein sequence ORF2 (FADα) - B. lata Wild typeMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 3 Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRTPRWEIVERFRNQPDKMDFMAPYPSSWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLVGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTADAIGIPRPEITYAIDDYVKRGAAHIREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 4 Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLFNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPNKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 5 Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 6 Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVALTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 7 Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGAANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPRPENCIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 8 Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLTDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSHIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 9 Protein sequence ORF2 (FADα) - B. cepacia F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 10 Protein sequence ORF2 (FADα) - B. cepacia F406D mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCDHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 11 Protein sequence ORF2 (FADα) - B. cepacia F406H mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCHHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 12 Protein sequence ORF2 (FADα) - B. cepacia F406M mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCMHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 13 Protein sequence ORF2 (FADα) - B. cepacia F406E mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCEHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 14 Protein sequence ORF2 (FADα) - B. cepacia F406S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCSHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 15 Protein sequence ORF2 (FADα) - B. cepacia F406T mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCTHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 16 Protein sequence ORF2 (FADα) - B. cepacia F406Y mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCYHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 17 Protein sequence ORF2 (FADα) - B. cepacia F406N mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCNHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 18 Protein sequence ORF2 (FADα) - B. cepacia F406Q mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCQHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 19 Protein sequence ORF2 (FADα) - B. cepacia F406C mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCCHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 20 Protein sequence ORF2 (FADα) - B. cepacia F406G mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCGHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 21 Protein sequence ORF2 (FADα) - B. cepacia F406P mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCPHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 22 Protein sequence ORF2 (FADα) - B. cepacia F406A mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCAHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 23 Protein sequence ORF2 (FADα) - B. cepacia F406V mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCVHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 24 Protein sequence ORF2 (FADα) - B. cepacia F406I mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCIHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 25 Protein sequence ORF2 (FADα) - B. cepacia F406W mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCWHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQID NO: 26 Protein sequence ORF2 (FADα) - B. cepacia N474H mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPHNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 27 Protein sequence ORF2 (FADα) - B. cepacia N474L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPLNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 28 Protein sequence ORF2 (FADα) - B. cepacia N474S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPSNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 29 Protein sequence ORF2 (FADα) - B. cepacia N474V mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPVNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 30 Primer used for random mutagenesis via error prone PCR methodCCAGGCAAATTCTGTTTTATCAGACC SEQ ID NO: 31 Primer used for randommutagenesis via error prone PCR method CAAGCTGGAGACCGTTTAAACTC SEQ IDNO: 32 Primer used for random mutagenesis via error prone PCR methodCGCTATTCAGATCCTCTTCTGAGATG SEQ ID NO: 33 Primer used for randommutagenesis via error prone PCR method GCTTCTGCGTTCTGATTTAATCTG SEQ IDNO: 34 Primer used for random mutagenesis via error prone PCR methodGGTCGTGGTCGGATCCGGTGTGGCAGGTGCTATTGTG SEQ ID NO: 35 Primer used forrandom mutagenesis via error prone PCR method CGTTCTTATTGCCCGAATAAACCSEQ ID NO: 36 Primer used for random mutagenesis via error prone PCRmethod CGAAGAAGCCCTGATGTTTGG SEQ ID NO: 37 Primer used for randommutagenesis via error prone PCR method GAAGCATGGTATCTGGGCATTGTTG Boldtext indicates mutations relative to wild-type within the amino acidsequence

TABLE 3 Protein sequences of FAD-GDHya from B. cepacia (SEQ ID NO: 38,SEQ ID NOs: 40-66) and B. lata (SEQ ID NO: 39). SEQ ID NO: 38 Proteinsequence ORF2 (FADα) - B. cepacia Wild typeMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 39Protein sequence ORF2 (FADα) B. lata Wild typeMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFFIVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 40Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRTPRWEIVERFRNQPDKMDFMAPYPSSWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLVGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 41Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLFNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRTVPNKTATDATGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 42Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 43Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVALTIAALALRMSDT LKKEV SEQ ID NO: 44Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGAANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPRPENCIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 45Protein sequence ORF2 (FADα) - B. cepacia mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLTDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSHIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 46Protein sequence ORF2 (FADα) - B. cepacia F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 47Protein sequence ORF2 (FADα) - B. cepacia F406D mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCDHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 48Protein sequence ORF2 (FADα) - B. cepacia F406H mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCHHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 49Protein sequence ORF2 (FADα) - B. cepacia F406M mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCMHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 50Protein sequence ORF2 (FADα) - B. cepacia F406E mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCEHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 51Protein sequence ORF2 (FADα) - B. cepacia F406S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCSHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 52Protein sequence ORF2 (FADα) - B. cepacia F406T mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCTHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 53Protein sequence ORF2 (FADα) - B. cepacia F406Y mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCYHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 54Protein sequence ORF2 (FADα) - B. cepacia F406N mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCNHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 55Protein sequence ORF2 (FADα) - B. cepacia F406Q mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCQHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 56Protein sequence ORF2 (FADα) - B. cepacia F406C mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCCHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 57Protein sequence ORF2 (FADα) - B. cepacia F406G mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCGHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 58Protein sequence ORF2 (FADα) - B. cepacia F406P mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCPHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 59Protein sequence ORF2 (FADα) - B. cepacia F406A mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCAHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 60Protein sequence ORF2 (FADα) - B. cepacia F406V mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCVHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 61Protein sequence ORF2 (FADα) - B. cepacia F406I mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCIHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 62Protein sequence ORF2 (FADα) - B. cepacia F406W mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCWHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 63Protein sequence ORF2 (FADα) - B. cepacia N474H mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPHNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 64Protein sequence ORF2 (FADα) - B. cepacia N474L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPLNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 65Protein sequence ORF2 (FADα) - B. cepacia N474S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPSNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 66Protein sequence ORF2 (FADα) - B. cepacia N474V mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPVNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV

TABLE 4 Protein sequences of FAD-GDHya for B. lata mutants withremovable 6x-His tag. SEQ ID Sequence SEQ ID NO: 67 Protein sequenceORF2 (FADα) - B. lata F406A mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCAHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 68 Protein sequence ORF2 (FADα) - B. lata F406C mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCCHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 69 Protein sequence ORF2 (FADα) - B. lata F406D mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCDHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 70 Protein sequence ORF2 (FADα) - B. lata F406E mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCEHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 71 Protein sequence ORF2 (FADα) - B. lata F406G mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCGHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 72 Protein sequence ORF2 (FADα) - B. lata F406H mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCHHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 73 Protein sequence ORF2 (FADα) - B. lata F406I mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCIHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 74 Protein sequence ORF2 (FADα) - B. lata F406K mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCKHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 75 Protein sequence ORF2 (FADα) - B. lata F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 76 Protein sequence ORF2 (FADα) - B. lata F406M mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCMHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 77 Protein sequence ORF2 (FADα) - B. lata F406N mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCNHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 78 Protein sequence ORF2 (FADα) - B. lata F406Q mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCQHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 79 Protein sequence ORF2 (FADα) - B. lata F406S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCSHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 80 Protein sequence ORF2 (FADα) - B. lata F406P mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCPHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 81 Protein sequence ORF2 (FADα) - B. lata F406V mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCVHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 82 Protein sequence ORF2 (FADα) - B. lata F406R mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCRHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 83 Protein sequence ORF2 (FADα) - B. lata F406T mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCTHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 84 Protein sequence ORF2 (FADα) - B. lata F406W mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCWHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQID NO: 85 Protein sequence ORF2 (FADα) - B. lata F406Y mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCYHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH*

TABLE 5 Protein sequences of FAD-GDHya for B. lata mutants. SEQ IDSequence SEQ ID NO: 86 Protein sequence ORF2 (FADα) - B. lata F406Amutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCAHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 87Protein sequence ORF2 (FADα) - B. lata F406C mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCCHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 88Protein sequence ORF2 (FADα) - B. lata F406D mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCDHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 89Protein sequence ORF2 (FADα) - B. lata F406E mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCEHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 90Protein sequence ORF2 (FADα) - B. lata F406G mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCGHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 91Protein sequence ORF2 (FADα) - B. lata F406H mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCHHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 92Protein sequence ORF2 (FADα) - B. lata F406I mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCIHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 93Protein sequence ORF2 (FADα) - B. lata F406K mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCKHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 94Protein sequence ORF2 (FADα) - B. lata F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 95Protein sequence ORF2 (FADα) - B. lata F406M mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCMHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 96Protein sequence ORF2 (FADα) - B. lata F406N mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCNHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 97Protein sequence ORF2 (FADα) - B. lata F406Q mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCQHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 98Protein sequence ORF2 (FADα) - B. lata F406S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCSHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 99Protein sequence ORF2 (FADα) - B. lata F406P mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCPHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 100Protein sequence ORF2 (FADα) - B. lata F406V mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCVHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO:101 Protein sequence ORF2 (FADα) - B. lata F406R mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCRHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO:102 Protein sequence ORF2 (FADα) - B. lata F406T mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCTHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO:103 Protein sequence ORF2 (FADα) - B. lata F406W mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCWHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO:104 Protein sequence ORF2 (FADα) - B. lata F406Y mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCYHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG

TABLE 6 Protein sequences according to some embodiments of the presentinvention. SEQ ID Sequence SEQ ID NO: 105 Protein sequence ORF2 (FADα) -N215D MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG D NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 106 Protein sequence ORF2 (FADα) - N215GMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG G NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 107 Protein sequence ORF2 (FADα) - N215HMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG H NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 108 Protein sequence ORF2 (FADα) - N215TMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG T NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO. 109 Protein sequence ORF2 (FADα) - N215YMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG Y NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 110 Protein sequence ORF2 (FADα) - B. cepacia N177S, N215S,F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRTPRWEIVERFRNQPDKMDFMAPYPSSWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSF S EQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG S NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPRILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGR GPQEMTSLVGFRDGP LRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNDHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 111 Protein sequence ORF2 (FADα) - B. lata N177S, N215S, F353L,F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSF S EQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCG S NNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGP LRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDC LHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 112 Protein sequence ORF2 (FADα) - B. lata N215S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 113 Protein sequence ORF2 (FADα) - B. lata N215T mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGT NNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 114 Protein sequence ORF2 (FADα) - B. cepacia N177S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSF S EQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 115 Protein sequence ORF2 (FADα) - B. lata N177S mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSF S EQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 116 Protein sequence ORF2 (FADα) - B. cepacia F353L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGP LRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKPDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 117 Protein sequence ORF2 (FADα) - B. lata F353L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGP LRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 118 Protein sequence ORF2 (FADα) - B. cepacia N177S, F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSF S EQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 119 Protein sequence ORF2 (FADα) - B. lata N177S, F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSF S EQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDC LHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 120 Protein sequence ORF2 (FADα) - B. cepacia F353L, F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGP LRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 121 Protein sequence ORF2 (FADα) - B. lata F353L, F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPLRANEAAKKIHLSNMSRINQETQKIFKGGKLMKPEELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 122 Protein sequence ORF2 (FADα) - B. cepacia N215S, F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG S NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 123 Protein sequence ORF2 (FADα) - B. lata N215S, F406L mutantMADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGS NNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDC LHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 124 Protein sequence ORF2 (FADα) - B. cepacia N177S, F353L, F406Lmutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSF S EQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGP LRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 125 Protein sequence ORF2 (FADα) - B. lata N177S, F353L, F406Lmutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSF S EQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGP LRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDC LHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 126 Protein sequence ORF2 (FADα) - B. cepacia N177S, N215S, F406Lmutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSF S EQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG S NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 127 Protein sequence ORF2 (FADα) - B. lata N177S, N215S, F406Lmutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSF S EQTIKSALNGYDPKFHVVTEPVARNSRPYDGRPTCCG S NNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDC LHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 128 Protein sequence ORF2 (FADα) - B. cepacia N215S, F353L, F406Lmutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRWEIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQRAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKFHVVTEPVARNSRPYDGRPTCCG S NNCMPICPIGAMYNGIVHVEKAERAGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGP LRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDC LHEILPQPENRIVPSKTATDAIGIPRPEITYAIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQID NO: 129 Protein sequence ORF2 (FADα) - B. lata N215S, F353L, F406Lmutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQRAEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGS NNCMPICPIGAMYNGIVHVEKAEQAGAKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGP LRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDC LHEILPQPENRIVPSKTATDAVGIPRPEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH

FIGS. 19A-B show some aspects of some embodiments of the presentinvention. FIG. 19A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215G to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 19B shows the non-linearfit of the data shown in FIG. 19A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 20A-B show some aspects of some embodiments of the presentinvention. FIG. 20A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215H to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 20B shows the non-linearfit of the data shown in FIG. 20A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 21A-B show some aspects of some embodiments of the presentinvention. FIG. 21A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215T to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 21B shows the non-linearfit of the data shown in FIG. 21A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 22A-B show some aspects of some embodiments of the presentinvention. FIG. 22A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215D to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 22B shows the non-linearfit of the data shown in FIG. 22A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 23A-B show some aspects of some embodiments of the presentinvention. FIG. 23A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215Y to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 23B shows the non-linearfit of the data shown in FIG. 23A, from which K_(m) (k) and V_(max) havebeen calculated.

FIGS. 24A-B show some aspects of some embodiments of the presentinvention. FIG. 24A shows electrochemical data representing the currentresponse of the biosensor comprising of FAD-GDHα N215S to variousglucose concentrations (shown as a square) and the linear fit across thedata range is represented by the R² value. FIG. 24B shows the non-linearfit of the data shown in FIG. 24A, from which K_(m) (k) and V_(max) havebeen calculated.

FIG. 25 shows electrochemistry data in connection with the wild typeFAD-GDHα protein.

FIG. 26 shows a table of electrochemistry data of the embodiments of thecomposition of the present invention. Mutations in position 406 provideimproved linearity over the entire range of physiological range:F406-S/C/T/V/Y/N/P/L/G/A/I/D/E.

FIG. 27 shows embodiments of the composition of the present invention.Mutations in position 406 that provide improved selectivity of glucose:F406-S/C/T/M/V/Y/N/P/L/G/Q/A/I/D/H/E. F406W provides an example of asubstitution that reduces the enzyme selectivity towards glucose.Mutations in position 215 provide improved linearity over the entirerange of physiological range: N215-G/H/T/D/Y/S.

An exemplary embodiment of the FAD-GDHα composition of the presentinvention is shown in FIGS. 25A and 25B, showing a graphicalrepresentation of the electrochemical response of a mutated FAD-GDHα(N177S, N215S, F353L, F406L) to various substrates. FIG. 25A showselectrochemical data representing the current response of the biosensorto various glucose concentrations (shown as a rhombus), maltoseconcentrations (shown as a triangle) and xylose (shown as a rectangle)concentrations (substrate solution were supplemented with 1 mMbenzoquinone). R² represents a linear fit of the glucose data. FIG. 25Bshows a biosensor response to glucose and the non-linear fit throughwhich apparent Km (k) and Imax have been calculated based onMichaelis-Menten equation:

$\begin{matrix}{I = \frac{{Imax}\lbrack S\rbrack}{{Km} + \lbrack S\rbrack}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Whereas “I” is the current, S is the substrate concentration, Imax isthe maximum current and the Km is the apparent Michaelis constant.

FIG. 26A shows an exemplary embodiment of a bioelectrochemical responseof an FAD-GDHα mutant (N177S, N215S, F353L, F406L) of the presentinvention, varying concentrations of glucose (shown as a rhombus). FIG.26B shows a bioelectrochemical response of an FAD-GDHα mutant (N177S,N215S, F353L, F406L) of the present invention to glucose and thenon-linear fit through which K_(m) (k) and V_(max) have been calculated.The enzyme activity of FAD-GDHα (N177S, N215S, F353L, F406L) wasdetermined by immobilizing FAD-GDHα (N177S, N215S, F353L, F406L) to acarbon electrode by an electropolymerization method without the additionof an electron mediator.

FIG. 27A shows an exemplary embodiment of the present invention, showinga biochemical response of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), maltose (shownas a triangle), an xylose (shown as a rectangle). FIG. 27B shows thebiochemical response of FAD-GDHα (N177S, N215S, F353L, F406L) to glucoseand the non-linear fit through which K_(m) (k) and V_(max) have beencalculated. FAD-GDHα (N177S, N215S, F353L, F406L) activity wasdetermined by monitoring a decrease of dichlorophenolindophenol (DCIP)signal at OD₆₀₀ (Epoch Microplate Spectrophotometer, Biotek).

FIGS. 28A and 28B, show an electrochemical response to varioussubstrates. In an exemplary embodiment, FIG. 28A shows electrochemicaldata representing the current response of the biosensor to variousglucose concentrations (shown as a rhombus), maltose concentrations(shown as a triangle) and xylose (shown as a rectangle) concentrations.FIG. 28B shows a biosensor response to glucose and the non-linear fitthrough which apparent K_(m) (k) and V_(max) have been calculated. In anexemplary embodiment, wild type FAD-GDHα was immobilized to a carbonelectrode via an electropolymerization method as described previously.R² represents a linear fit of the glucose data. FIG. 28B showsK_(mapp)=0.85 mM and Imax=1218.5 nA.

FIG. 29A shows biochemical responses of wild type FAD-GDHα (w.t.),varying concentrations of glucose (rhombus), maltose (triangle) andxylose (rectangle). FIG. 29B shows a biochemical response of wild typeFAD-GDHα to glucose and the non-linear fit through which K_(m) (k) andV_(max) have been calculated. The enzyme activity of wild type FAD-GDHαwas determined by monitoring the decrease of Dichlorophenolindophenol(DCIP) signal at OD₆₀₀.

Biochemical and Electrochemical comparison between a mutant according tosome embodiments of the present invention and FAD-GDHα (w.t.), shown inFIGS. 27A and 27B as compared with FIGS. 29A and 29B, demonstrates thatthe mutant increased in specificity and activity while FIGS. 28A and 28Bdemonstrate the improvement in bioelectrochemical linearity behavior.Thus, these results distinguish the mutant for glucose-measurementsapplications over the wild type enzyme.

FIG. 30 is a table showing the results obtained from the biochemical andelectrochemical experiments characterizing FAD-GDHα (N177S, N215S,F353L, F406L) where: K_(m) is the Michaelis constant, K_(cat) is theenzyme's catalytic constant, K_(cat)/K_(m) is the catalytic efficiency,glucose/xylose is the ratio of K_(catglu)/K_(catxyl), BEC linearity isthe R² of the linear fit of the bioelectrochemical experiment, 120 isthe current flux (nA/cm²) measured when 20 mM of glucose was tested,Xylose selectivity is the ratio of I20_(glu)/I20_(xyl), Maltoseselectivity is the ratio of I20_(glu)/I20_(mal), pos1-4, the mutatedamino acid number.

FIG. 34A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized withpolypyrrole, and benzoquinone is used as a mediator to obtain a singlemeasurement. FIG. 34B shows the non-linear fit (red line) of the datarepresented in FIG. 34A. V_(max) refers to the maximum current flux, Krefers to the apparent K_(m) value extracted from the Michaelis mentenequation.

The screen printed electrode (SPE) was activated by overlaying it withsaturated potassium bicarbonate solution and applying 1.2V for 3minutes. The electrode was washed 3 times with DDW and overlaid withimmobilization solution containing 1.2 mg/ml of enzyme and 0.25 MPyrrole-KCl solution. Electropolymerization was applied via CA 20 pulsesof 0.65V, 1 second each with 5 second intervals between each pulse.Glucose sensing was conducted by dipping the immobilized enzyme SPE intoa PBS solution, followed by step-wise pipetting of concentrated glucosesolution to reach the depicted final glucose solution and by applying0.2V for 5 minutes at each addition point

FIG. 35A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized withpolypyrrole, to obtain a single measurement via direct electrontransfer. FIG. 35B shows the non-linear fit (red line) of the datarepresented in FIG. 35A. V_(max) refers to the maximum current flux, Krefers to the apparent K_(m) value extracted from the Michaelis mentenequation.

The screen printed electrode (SPE) was activated by overlaying it withsaturated potassium bicarbonate solution and applying 1.2V for 3minutes. The electrode was washed 3 times with DDW and overlaid withimmobilization solution containing 1.2 mg/ml of enzyme and 0.25 MPyrrole-KCl solution. Electropolymerization was applied via CA 20 pulsesof 0.65V, 1 second each with 5 second intervals between each pulse.Glucose sensing was conducted by dipping the immobilized enzyme SPE intoa PBS solution, followed by step-wise pipetting of concentrated glucosesolution to reach the depicted final glucose solution and by applying0.2V for 5 minutes at each addition point.

FIG. 36A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized with PEDOT,to obtain a single measurement via direct electron transfer. FIG. 36Bshows the non-linear fit (red line) of the data represented in FIG. 36A.V_(max) refers to the maximum current flux, K refers to the apparentK_(m) value extracted from the Michaelis menten equation.

The printed electrode (SPE) was activated by overlaying it withsaturated potassium bicarbonate solution and applying 1.2V for 3minutes. The electrode was washed 3 times with DDW and overlaid withimmobilization solution containing 1 mg/ml of enzyme and 0.01 M EDOT/PSSsolution (final concentration). Electropolymerization was applied viacycles of CV ranging 0.2-0.85V with a scanning speed of 0.05V/Sec. Theimmobilized electrode was then washed with DDW and glucose sensing wasconducted by dipping the immobilized enzyme SPE into a PBS solution,followed by step-wise pipetting of concentrated glucose solution toreach the depicted final glucose solution and by applying 0.3V for 5minutes at each addition point.

FIG. 37A shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L) tovarying concentrations of glucose (shown as a rhombus), usingscreen-printed electrodes according to some embodiments of the presentinvention, where the mutant FAD-GDH protein is immobilized with grapheneoxide, to obtain a single measurement via direct electron transfer. FIG.37B shows the non-linear fit (red line) of the data represented in FIG.37A. V_(max) refers to the maximum current flux, K refers to theapparent K_(m) value extracted from the Michaelis menten equation.

The printed electrode (SPE) was activated by overlaying it withsaturated potassium bicarbonate solution and applying 1.2V for 3minutes. The electrode was washed 3 times with DDW and overlaid withimmobilization solution containing 1 mg/ml of enzyme and 0.01 M EDOT/PSSsolution (final concentration). Electropolymerization was applied viacycles of CV ranging 0.2-0.85V with a scanning speed of 0.05V/Sec. Theimmobilized electrode was then washed with DDW and glucose sensing wasconducted by dipping the immobilized enzyme SPE into a PBS solution,followed by step-wise pipetting of concentrated glucose solution toreach the depicted final glucose solution and by applying 0.3V for 5minutes at each addition point.

FIGS. 38A and 38B shows an exemplary embodiment of the presentinvention, showing electrochemistry data of FAD-GDHα (N177S, N215S,F353L, F406L), using an electrode according to some embodiments of thepresent invention configured to measure glucose levels continuously, for20 hrs (FIG. 38A), or 64 hr (FIG. 38B). In this the mutant FAD-GDHprotein is immobilized with polypyrrole, to obtain a single measurementvia direct electron transfer.

SPE was activated by overlaying it with saturated potassium bicarbonatesolution and applying 1.2V for 3 minutes. The electrode was washed 3times with DDW and overlaid with immobilization solution containing 1.2mg/ml of enzyme and 0.25 M Pyrrole-KCl solution. Electropolymerizationwas applied via Chronoamperometry (CA) by applying 20 pulses of 0.65V, 1second each with 5 second intervals between each pulse. Glucose sensingwas conducted by dipping the immobilized enzyme SPE into a PBS solutioncontaining 5.5 mM glucose and by applying 0.2V for 20 or 64 hours (FIGS.38A and 38B, respectively).

FIG. 39 shows an exemplary embodiment of the present invention, showingelectrochemistry data of FAD-GDHα (N177S, N215S, F353L, F406L), using anelectrode according to some embodiments of the present inventionconfigured to measure glucose levels continuously, for 20 hrs. In thisembodiment, the mutant FAD-GDH protein is immobilized with PEDOT, toobtain a single measurement via direct electron transfer.

SPE was activated by overlaying it with saturated potassium bicarbonatesolution and applying 1.2V for 3 minutes. The electrode was washed 3times with DDW and overlaid with immobilization solution containing 1mg/ml of enzyme and 0.01 M EDOT/PSS solution (final concentration).Electropolymerization was applied via cycles of CV ranging 0.2-0.85Vwith a scanning speed of 0.05V/Sec. Electropolymerization was appliedvia Chronoamperometry (CA) by applying 20 pulses of 0.65V, 1 second eachwith 5 second intervals between each pulse. Glucose sensing wasconducted by dipping the immobilized enzyme SPE into a PBS solutioncontaining 5.5 mM glucose and by applying 0.3V for 18 hours.

FIG. 40 shows a table of electrochemistry data of the embodiments of theelectrodes of the present invention.

While a number of embodiments of the present invention have beendescribed, it is understood that these embodiments are illustrativeonly, and not restrictive, and that many modifications may becomeapparent to those of ordinary skill in the art. Further still, thevarious steps may be carried out in any desired order (and any desiredsteps may be added and/or any desired steps may be eliminated). Allpublications and other references mentioned herein are incorporated byreference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference.

Publications and references cited herein are not admitted to be priorart.

REFERENCES

-   Wang, Joseph (2008). Electrochemical Glucose Biosensors. Chem. Rev.    2008, 108, 814-825.-   Ferri, Stefano et al. (2011) Review of Glucose Oxidases and Glucose    Dehydrogenases: A Bird's Eye View of Glucose Sensing Enzymes.    Journal of Diabetes Science and Technology. Volume 5, Issue 5,    September 2011.

1. A mutated FAD-GDH protein, wherein the mutated FAD-GDHα protein ismutated from a wild-type first species to contain at least one pointmutation, wherein the mutated FAD-GDHα protein comprises:P(X)_(n=8)X⁴(X)_(n=16)V(X)_(n=6)RN(X)_(n=3)YDXRPXCXGX³NNCMP(X)_(n=1)CP(X)_(n=2)A(X)_(n=1)Y(X)_(n=1)G(X)_(n=6)A(X)_(n=2)AG(X)_(n=6)AVV(X)_(n=3)E(X)_(n=8-9)A(X)_(n=2)Y(X)_(n=1)D(X)_(n=5)HRV(X)_(n=5)V(X)_(n=2)A(X)_(n=3)E(X)_(n=2)K(X)_(n=4)S(X)_(n=5)P(X)_(n=1)G(X)_(n=2)N(X)_(n=4)GRN(X)_(n=1)MDH(X)_(n=4)V(X)_(n=1)F(X)_(n=6-7)W(X)_(n=1)GRGP(X)_(n=9)RDGXX⁵R(X)_(n=19)T(X)_(n=14)L(X)_(n=14)X²(X)_(n=1)(X)_(n=1)E(X)_(n=4)P(X)_(n=1)NR(X)_(n=3)S(X)_(n=4)D(X)_(n=2)G(X)_(n=7)Y(X)_(n=4)Y(X)_(n=32-35),wherein each X represents a wild-type amino acid residue of the firstspecies and n indicates the number of the wild-type amino acid residuesof the first species represented by a respective parenthetical at thatposition, wherein: a) X¹ is selected from the group consisting of X, S,C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L,H or V, then X² is D; b) X³ is selected from the group consisting of G,H, D, Y, S, and X; c) X⁴ is selected from the group consisting of S andX; and d) X⁵ is selected from the group consisting of S and X.
 2. Themutated FAD-GDHα protein of claim 1, wherein: a) X¹ is selected from thegroup consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, andE, wherein if X¹ is L, H or V, then X² is D; b) X³ is selected from thegroup consisting of G, H, D, Y, S, and X; c) X⁴ is selected from thegroup consisting of S and X; and d) X⁵ is selected from the groupconsisting of S and X.
 3. The mutated FAD-GDHα protein of claim 1,wherein: a) X¹ is selected from the group consisting of S, C, T, M, V,Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V, thenX² is D; b) X³ is selected from the group consisting of G, H, D, Y, S,and X; c) X⁴ is X; and d) X⁵ is X.
 4. The mutated FAD-GDHα protein ofclaim 1, wherein: a) X¹ is selected from the group consisting of S, C,T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, Hor V, then X² is D; b) X³ is X; c) X⁴ is X; and d) X⁵ is X.
 5. Themutated FAD-GDHα protein of claim 1, wherein: a) X¹ is X; b) X³ isselected from the group consisting of G, H, D, Y, S, and X; c) X⁴ is X;and d) X⁵ is X.
 6. The mutated FAD-GDHα protein of claim 1, wherein: a)X¹ is selected from the group consisting of S, C, T, M, V, Y, N, P, L,G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V, then X² is D; b)X³ is selected from the group consisting of G, H, D, Y, and S; c) X⁴ isS; and d) X⁵ is X.
 7. The mutated FAD-GDHα protein of claim 1, wherein:a) X¹ is selected from the group consisting of S, C, T, M, V, Y, N, P,L, G, Q, A, I, D, W, H, and E, wherein if X¹ is L, H or V, then X² is D;b) X³ is selected from the group consisting of G, H, D, Y, and S; c) X⁴is X; and d) X⁵ is S.
 8. The mutated FAD-GDHα protein of claim 1,wherein: a) X¹ is selected from the group consisting of S, C, T, M, V,Y, N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹ is L, H or V, thenX² is D; b) X³ is X; c) X⁴ is S; and d) X⁵ is S.
 9. The mutated FAD-GDHαprotein of claim 1, wherein: a) X¹ is selected from the group consistingof S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, or E, wherein if X¹is L, H or V, then X² is D; b) X³ is selected from the group consistingof G, H, D, Y, and S; c) X⁴ is S; and d) X⁵ is S.
 10. The mutatedFAD-GDHα protein of claim 1, wherein the amino acid sequence of themutated FAD-GDHα protein comprises the amino acid sequence set forth inany one of SEQ ID NOS: 9-29, SEQ ID NOS: 46-104, or SEQ ID NOS: 105-129.11. The mutated FAD-GDHα protein of claim 1, wherein the biochemicalactivity is increased at least 10% compared to a non-mutated FAD-GDHαprotein from the wild type first species, wherein the selectivity forglucose is increased at least 10% compared to a non-mutated FAD-GDHαprotein from the wild type first species or wherein the linearity ofcurrent as a function of glucose concentration is increased at least 10%compared to a non-mutated FAD-GDHα protein from the wild type firstspecies.
 12. (canceled)
 13. (canceled)
 14. An enzyme electrode,configured to measure the amount of glucose in a physiological fluid,comprising the mutated FAD-GDHα of claim 1 immobilized onto theelectrode, wherein the mutated FAD-GDHα is configured to catalyzeglucose in the physiological fluid and produce electrons that aretransferred to the electrode thereby generating an electrical current,wherein the intensity of the electrical current is indicative of thelevel of glucose in the physiological fluid.
 15. (canceled)
 16. Theenzyme electrode of claim 14, wherein the enzyme electrode is configuredto perform a single measurement.
 17. The enzyme electrode of claim 14,wherein the enzyme electrode is incorporated into a glucose test strip.18. The enzyme electrode of claim 14, wherein the mutated FAD-GDH isimmobilized on the electrode in a conductive matrix or by chemicalwiring.
 19. The enzyme electrode of claim 18, wherein the conductivematrix is selected from a group consisting of carbon paste, graphitepaste, and graphene oxide or wherein the conductive matrix is aconductive polymer and wherein the conductive polymer is selected fromthe group consisting of PEDOT and Polypyrrol.
 20. (canceled) 21.(canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)26. (canceled)
 27. (canceled)
 28. The enzyme electrode of claim 14,wherein the enzyme electrode, configured to measure the amount ofglucose in a physiological fluid, comprising the mutated FAD-GDHαprotein immobilized onto the electrode further comprises at least onesubunit selected from the group consisting of: wild-type FAD-GDHβsubunit, and a wild-type FAD-GDHγ subunit.
 29. The enzyme electrode ofclaim 14, wherein the enzyme electrode is incorporated into a biosensorconfigured for subcutaneous continuous glucose measurement, wherein thebiosensor is configured to continually measure the amount of glucose ina subject.
 30. The enzyme electrode of claim 29, wherein the biosensorcomprises the mutated FAD-GDH immobilized onto at least one enzymeelectrode, wherein the mutated FAD-GDH is configured to catalyze glucosein the subject and generate electrons that are transferred to theelectrode and generate electrical current, wherein the intensity of theelectrical current is indicative of the level of glucose in the subject.31. (canceled)
 32. The enzyme electrode of claim 30, wherein the enzymeelectrode is configured to perform a continuous measurement for up totwo weeks.
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)37. (canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled) 41.(canceled)
 42. (canceled)